Methods of treating anaemia

ABSTRACT

The invention relates to human targets of interest (TOI), anti-TOI ligands, kits compositions and method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/211,178, filed Jul. 15, 2016, which is a continuation of U.S. patent application Ser. No. 15/041,514, filed Feb. 11, 2016, now U.S. Pat. No. 9,428,578, which is a continuation of U.S. patent application Ser. No. 14/978,183, filed Dec. 22, 2015, now U.S. Pat. No. 9,394,568, which is a continuation of U.S. patent application Ser. No. 14/868,952, filed Sep. 29, 2015, now U.S. Pat. No. 9,303,089, which is a continuation of U.S. patent application Ser. No. 14/731,727, filed Jun. 5, 2015, now U.S. Pat. No. 9,187,562, which is a continuation of U.S. patent application Ser. No. 14/665,579, filed Mar. 23, 2015, now U.S. Pat. No. 9,139,648, which is a continuation-in-part of U.S. patent application Ser. No. 14/331,730, filed Jul. 15, 2014, now U.S. Pat. No. 9,914,769, the entire contents of which are herein incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on created on created on Jul. 6, 2021, is named 720027_SA9-639CIPCON7_ST25.txt and is 624,021 bytes in size.

TECHNICAL FIELD

The technology described herein relates to ligands, e.g., antibodies for the treatment of disease.

BACKGROUND

It is recognized that individual humans differ in their sequence and recently several individuals have had their genomes sequenced, for instance James Watson and Craig Venter. Comparison of the genome sequence of individuals has revealed differences in their sequences in both coding and non-coding parts of the genome. Some of these variations in humans are significant and contribute to phenotypic differences between individuals. In extreme cases these will result in genetic disease. The 1000 Genomes Project has the objective of cataloguing sequences in the human genome, involving sequencing the genomes of a very large sampling of individuals from diverse art-recognized human ethnic populations.

SUMMARY

Through the application of human genetic variation analysis and rationally-designed sequence selection the present invention provides for improved human patient diagnosis and therapy. Importantly, the invention enables tailored medicines that address individual human patient genotypes or phenotypes.

The inventor's analysis of large numbers of naturally-occurring genomic human TOI sequences reveals that there is significant variation across diverse human populations and provides for the ability for correlation between individual human patients and tailored medical and diagnostic approaches addressing the target. The technical applications of these findings, as per the present invention, thus contribute to better treatment, prophylaxis and diagnosis in humans and provides for patient benefit by enabling personalized medicines and therapies. This provides advantages of better prescribing, less wastage of medications and improved chances of drug efficacy and better diagnosis in patients.

Furthermore, the inventor surprisingly realised that some rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting such rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention and better serving patients in those populations.

With this, the inventor realised that there is significant industrial and medical application for the invention in terms of guiding the choice of an anti-TOI ligand for administration to human patients for therapy and/or prophylaxis of TOI-mediated or associated diseases and conditions. In this way, the patient receives drugs and ligands that are tailored to their needs—as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

To this end, the invention provides:—

In a First Configuration

A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising

-   a. Administering to the human an anti-TOI ligand to target a TOI     variant in the human and treat or prevent said disease or condition,     wherein the TOI in said human is encoded by a nucleotide sequence     having a cumulative human allele frequency of less than 50% and/or     wherein the TOI in said human is encoded by a nucleotide sequence     having a total human genotype frequency of less than 50%;

wherein

-   b. Before step (a) said human has been or is genotyped as positive     for said nucleotide sequence or phenotyped as positive for said TOI     variant.

In a Second Configuration

A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI         variant in the human and treat or prevent said disease or         condition, wherein the TOI in said human is encoded by a         nucleotide sequence having a cumulative human allele frequency         of less than 50% and/or wherein the TOI in said human is encoded         by a nucleotide sequence having a total human genotype frequency         of less than 50%; wherein     -   b. before step (a) the ligand has been or is determined as         capable of binding to said TOI variant.

In a Third Configuration

A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising

-   a. Administering to the human an anti-TOI ligand to target a TOI     variant in the human and treat or prevent said disease or condition,     wherein the TOI in said human is a variant encoded by a nucleotide     sequence having a cumulative human allele frequency of more than 50%     and/or having a total human genotype frequency of more than 50%;     wherein -   b. Before step (a) said human has been or is genotyped as negative     for a variant nucleotide sequence having a cumulative human allele     frequency of less than 50% and/or having a total human genotype     frequency of less than 50%; or phenotyped as negative for a TOI     variant encoded by a nucleotide sequence having a cumulative human     allele frequency of less than 50% and/or having a total human     genotype frequency of less than 50%.

In a Fourth Configuration

An anti-human TOI ligand for use in a method of treating and/or preventing a TOI-mediated disease or condition in a human, wherein the TOI is present in humans as different polymorphic variants and wherein the genome of said human comprises a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, the method comprising administering the ligand to the human.

In a Fifth Configuration

A ligand that binds a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, for use in a method comprising the step of using the ligand to target said TOI in a human to treat and/or prevent a disease or condition mediated by TOI, the method comprising administering the ligand to the human.

In a Sixth Configuration

A pharmaceutical composition or kit for treating or preventing a condition or disease mediated by a TOI.

In a Seventh Configuration

A method of producing an anti-human TOI antibody binding site, the method comprising obtaining a plurality of anti-TOI antibody binding sites, screening the antibody binding sites for binding to a TOI comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOI-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody binding site that binds in the screening step.

In a Eighth Configuration

A method of producing an anti-human TOI antibody, the method comprising immunising a non-human vertebrate (eg, a mouse or a rat) with a TOI comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOI-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody that binds a TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, and optionally producing a TOI-binding fragment or derivative of the isolated antibody.

In a Ninth Configuration

A kit for TOI genotyping a human, wherein the kit comprises a nucleic acid comprising a nucleotide sequence that specifically hybridises to a TOI nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or an RNA transcript thereof; and/or the nucleic acid comprises a nucleotide sequence that comprises at least 10 contiguous nucleotides of a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or is an antisense sequence thereof.

In a Tenth Configuration

Use of an anti-TOI ligand that binds a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for treating and/or preventing a TOT-mediated disease or condition in a human whose genome comprises a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

In a Eleventh Configuration

Use of an anti-TOI ligand that binds a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for targeting said TOI in a human to treat and/or prevent a disease or condition mediated by TOI.

In a Twelfth Configuration

A method of targeting a TOI for treating and/or preventing a TOT-mediated disease or condition in a human, the method comprising administering an anti-TOI ligand to a human comprising a TOI nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, whereby a TOI encoded by said nucleotide sequence is targeted.

In a Thirteenth Configuration

A method of TOI genotyping a nucleic acid sample of a human, the method comprising identifying in the sample the presence of a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

In a Fourteenth Configuration

A method of TOI typing a protein sample of a human, the method comprising identifying in the sample the presence of a TOI amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

In an example, the TOI is a human TOI selected from the group consisting of PCSK9, VEGF-A and IL6 receptor. In an example, the TOI is human IL4Ra, PDGF-B, PDGFR-B or Ang-2.

A Fifteenth Configuration provides a ligand, method, use, kit or composition of the invention, wherein

-   (i) the ligand (eg, antibody or fragment) comprises     -   (a) a variable domain that is encoded by a human V region         nucleotide sequence, wherein the V nucleotide sequence is         derived from recombination of human VH, D and JH gene segments         or human VL and JL gene segments; or     -   (b) a constant region domain encoded by a C region gene segment;     -   Wherein a first gene segment of said gene segments of (a), or         said C region gene segment of (b) comprises a first single         nucleotide polymorphism (SNP) encoding a first amino acid         polymorphism; and -   (ii) the genome of said human comprises said first SNP or wherein     said human expresses (a′) an antibody variable domain comprising     said first amino acid polymorphism or (b′) an antibody constant     domain comprising said first amino acid polymorphism.

A Sixteenth Configuration provides the ligand, method, use, kit or composition of the invention, wherein the ligand comprises or consists of an antibody or fragment that comprises a human antibody variable domain derived from the recombination of a human V gene segment and a human J gene segment (and optionally a human D gene segment when the variable domains are VH domains); and wherein the genome of the human comprises said human V gene segment and/or the human expresses antibodies comprising antibody variable domains derived from the recombination of said human V gene segment and a human J gene segment (and optionally a human D gene segment).

A Sixteenth Configuration provides the ligand, method, use, kit or composition of the invention, wherein the ligand (eg, comprising or consisting of an antibody or fragment or an Fc-fused human PCSK9 receptor) comprises a human heavy chain constant domain encoded by a first constant region nucleotide sequence; and wherein the genome of the human comprises a heavy chain constant region nucleotide sequence that is identical to said first constant region nucleotide sequence and/or the human expresses antibodies comprising said human constant domain.

A Seventeenth Configuration provides the ligand, method, use, kit or composition of the invention, wherein the ligand (eg, comprising or consisting of an antibody or fragment or an Fc-fused TOI receptor) comprises a human gamma-I heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein the genome of the human comprises a gamma-1 heavy chain constant region nucleotide sequence that encodes such an Asp or Leu or the human expresses antibodies comprising human gamma-1 constant regions comprising such an Asp or Leu.

A Eighteenth Configuration provides the ligand, method, use, kit or composition of the invention, wherein the ligand (eg, comprising or consisting of an antibody or fragment or an Fc-fused TOI receptor) comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44; and wherein the genome of the human comprises a gamma-2 heavy chain constant region nucleotide sequence that encodes such a selected amino acid or the human expresses antibodies comprising human gamma-2 constant regions comprising such a selected amino acid.

A Nineteenth Configuration provides the ligand, method, use, kit or composition of the invention, wherein the ligand (eg, comprising or consisting of an antibody or fragment or an Fc-fused TOI receptor) comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50; and wherein the genome of the human comprises a kappa light chain constant region nucleotide sequence that encodes such a Val or Cys or the human expresses antibodies comprising human kappa light chain constant regions comprising such a Val or Cys.

A Twentieth Configuration provides the ligand, method, use, kit or composition of the invention, wherein the ligand comprises or consists of an antibody or fragment, wherein the antibody or fragment comprises a VH domain that is derived from the recombination of a human VH gene segment, a human D gene segment and a human JH gene segment, wherein the VH gene segment is selected from the group consisting of (i) IGHV1-18*01 and the genome of the human comprises a human IGHV1-18*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGHV1-18*01; or (ii) IGVH1-46*01 and the genome of the human comprises a human IGHV1-46*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGHV1-46*01.

A Twenty-First Configuration provides the ligand, method, use, kit or composition of the invention, wherein the ligand comprises or consists of an antibody or fragment, wherein the antibody or fragment comprises a VL domain that is derived from the recombination of a human VL gene segment and a human JL gene segment, wherein the VL gene segment is selected from the group consisting of (i) IGKV4-1*01 and the genome of the human comprises a human IGKV4-1*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGKV4-1*01; (ii) IGLV2-14*01 and the genome of the human comprises a human IGLV2-14*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGLV2-14*01; or (iii) IGKV1-13*02 and the genome of the human comprises a human IGKV1-13*02 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGKV1-13*02.

A Twenty-Second Configuration provides a method of treating or reducing the risk of an IL4Ra-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. As explained further below, these amino acid variations are found in naturally-occurring IL-4Ra variants in humans found in many populations. Said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

A Twenty-Third Configuration provides a ligand (eg, an antibody or antibody fragment) for treating or reducing the risk of an IL4Ra-mediated disease or condition in a human in need thereof, the method comprising administering to said human said ligand, wherein the ligand specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

A Twenty-Fourth Configuration provides provides a method of targeting IL4Ra in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In an example, the human is suffering from or at risk of an IL4Ra-mediated disease or condition. In an example, the method treats or reduces the risk of an IL4Ra-mediated disease or condition in the human.

A Twenty-Fifth Configuration provides a ligand (eg, an antibody or antibody fragment) for targeting IL4Ra in a human, the method comprising administering to said human said ligand, wherein the ligand specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In an example, the human is suffering from or at risk of an IL4Ra-mediated disease or condition. In an example, the method treats or reduces the risk of an IL4Ra-mediated disease or condition in the human.

In an embodiment of any of the 21-25^(th) configurations, (i) the antibody or fragment comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and wherein (ii) said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

Additionally or alternatively, in an embodiment of any of the 21-25^(th) configurations, (i) the antibody or fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In a Twenty-sixth Configuration, described herein is a method of treating or reducing the risk of a Nav1.7-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G; wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The invention also provides a corresponding ligand, eg, antibody or antibody fragment, for use in such a method.

In some embodiments, said amino acid is selected from the group consisting of any one of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said disease or condition is a pain disease or condition. In some embodiments, said amino acid is selected from the group consisting of 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; optionally wherein said disease or condition is a pain disease or condition. In some embodiments, said amino acid is selected from the group consisting of 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K) and 1689X (wherein X is an amino acid other than W); optionally wherein said disease or condition is a pain disease or condition.

In some embodiments, the constant region gene segment comprised by said human is a germline gene segment. In some embodiments, the method further comprises, before said administering, selecting a human comprising said nucleotide sequence of (ii).

In some embodiments, the human has been determined to comprise the nucleotide sequence that encodes a Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G and/or a Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In some embodiments, the method further comprises the step of determining that the human comprises (a) the nucleotide sequence that encodes a Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G and/or (b) a Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, optionally, wherein the determining step is performed before administration of the antibody to the human. In some embodiments, the step of determining comprises assaying a biological sample from the human for a nucleotide sequence encoding a Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. In some embodiments, the assaying comprises contacting the biological sample with at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides of a nucleotide sequence encoding a Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or comprising an antisense sequence of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises a nucleotide sequence encoding said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby forming a complex when at least one nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is present; and detecting the presence or absence of the complex, wherein detecting the presence of the complex determines that the human comprises the Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. In some embodiments, the assaying comprises nucleic acid amplification and optionally one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification and/or wherein the assaying is performed in a multiplex format. In some embodiments, said biological sample comprises serum, blood, faeces, tissue, a cell, urine and/or saliva of said human.

In some embodiments, said human is indicated as heterozygous for a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, optionally, wherein said human is further indicated as comprising the nucleotide sequence of SEQ ID NO: 76, or said human is indicated as homozygous for a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In some embodiments, said human is or has been further determined to be substantially resistant to a pain or itching treatment. In some embodiments, said human is receiving or has received a pain or anti-itching treatment or has reduced responsiveness to a pain or itching treatment. In some embodiments, said disease or condition is a pain or itching disease or condition. In some embodiments, said disease or condition is a channelopathy or associated with a channelopathy; or is selected from the group consisting of primary erythermalgia (PE), paroxysmal extreme pain disorder (PEPD) and channelopathy-associated insensitivity to pain (CIP). In some embodiments, said human has been diagnosed with a pain or itching disease or condition.

In some embodiments, said ligand fragment treats or reduces the risk in said human of a pain or itching disease or condition.

In some embodiments, the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs6746030, rs3750904, rs58022607, rs4369876, rs13402180 and rs12478318.

In a Twenty-seventh Configuration, described herein is a method of treating or reducing the risk of a disease or condition mediated by VEGF-A in a human, the method comprising administering to said human an anti-VEGF-A ligand (eg, an anti-VEGF-A trap, antibody or antibody fragment) that specifically binds to a human VEGF-A that is expressed by a VEGF-A nucleotide sequence comprising a SNP selected from the group consisting of rs699947, rs833061, rs2010963, rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069, rs3025000 and rs1570360, wherein said human comprises a VEGF-A nucleotide sequence comprising said selected SNP. The invention also provides a corresponding ligand, eg, antibody or antibody fragment, for use in such a method.

In a Twenty-eighth Configuration, described herein is a method of treating or reducing the risk of a disease or condition mediated by VEGF-A in a human, the method comprising administering to said human an anti-VEGF-A ligand (eg, an anti-VEGF-A trap, antibody or antibody fragment) that specifically binds to a human VEGF-A, wherein the human comprises (i) an ARMS2 nucleotide sequence comprising a G at the position of SNP rs10490924; (ii) a CFH nucleotide sequence comprising a T at the position of SNP rs1061170 or an T at the position of rs3766404; or (iii) a VEGFR2 nucleotide sequence comprising SNP rs4576072 or rs6828477, wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an amino acid selected from the group consisting of an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising said selected amino acid. These SNPs are associated with improved responses following anti-VEGF-A treatment of an ocular disease or condition. The invention also provides a corresponding ligand, eg, antibody or antibody fragment, for use in such a method.

In a Twenty-ninth Configuration, described herein is a method of treating or reducing the risk of a VEGF-A mediated disease or condition in a human, the method comprising administering an anti-human VEGF-A ligand to a human, wherein the human comprises (i) a PDGF-B nucleotide sequence comprising a SNP selected from the group consisting of rs142404523 (ie, a C corresponding to position −776) and a C at the position of rs1800818 (ie, a C corresponding to position −735); or (ii) a PDGFR-B nucleotide sequence comprising a SNP selected from the group consisting of rs246395 (ie, a G corresponding to position 2601) and rs74943037 (ie, a T corresponding to position 1391). The invention also provides a corresponding ligand, eg, antibody or antibody fragment, for use in such a method.

In an example of any of the 27-29th configurations, the ligand comprises an antibody constant region (eg, an antibody Fc region). Optionally, the ligand comprises a human gamma-1 heavy chain constant region that comprises an amino acid selected from the group consisting of an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising said selected amino acid.

In an example of any of the 27-29th configurations the disease or condition is an ocular condition or a cancer; or angiogenesis or neovascularisation.

In an example of any of the 27-29th configurations the method further comprises antagonising PDGF-B in said human.

In an example of any of the 27-29th configurations the method further comprises antagonising angiopoietin-2 (Ang2) in said human.

In a Thirtieth configuration, the invention provides:—

A method of treating or reducing the risk of a disease or condition mediated by PD-L1 in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21.

For example, there is provided a method of treating or reducing the risk of a disease or condition mediated by PD-L1 in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21, wherein said human comprises a PD-L1 nucleotide sequence comprising said selected variation.

For example, there is provided a method of treating or reducing the risk of a cancer in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21, wherein said human comprises a PD-L1 nucleotide sequence comprising said selected variation.

For example, there is provided a method of treating or reducing the risk of an autoimmune disease or condition in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21, wherein said human comprises a PD-L1 nucleotide sequence comprising said selected variation.

For example, there is provided a method of treating or reducing the risk inflamatory disease or condition in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21, wherein said human comprises a PD-L1 nucleotide sequence comprising said selected variation.

In a Thirty-first configuration, the invention provides an anti-human PD-L1 ligand (eg, an antibody, antibody fragment or human PD-L1 trap) for use in a method of the Thirtieth configuration.

In a Thirty-second configuration the invention provides: A method of cancer immunotherapy in a human by targeting an immune cell TOI (eg, PD-1) in the human, the TOI being present in humans as a plurality of variants differing by one or more amino acid polymorphisms, the method comprising administering a ligand (eg, an antibody or antibody fragment) to the human, the ligand comprising first and second protein domains, wherein the first domain specifically binds a TOI variant comprising a first amino acid polymorphism, wherein the second domain comprises a second polymorphism, and wherein the human expresses (i) TOI comprising said first amino acid polymorphism; and (ii) protein domains comprising said second polymorphism, wherein the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein the second domain is comprised by said gamma-4 heavy chain constant region of the ligand.

This configuration also provides the following aspects:—

A method of cancer immunotherapy in a human by targeting PD-1 in the human, the method comprising administering an antibody or antibody fragment to the human, wherein the antibody or antibody fragment specifically binds a PD-1 encoded by a PD-1 nucleotide sequence comprising a SNP selected from the group consisting of the variations set out in Table 23, wherein the antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide sequence comprising said selected SNP.

A method of cancer immunotherapy in a human by targeting PD-1 in the human, the method comprising administering an antibody or antibody fragment to the human, wherein the antibody or antibody fragment specifically binds a PD-1 encoded by a PD-1 nucleotide sequence comprising a SNP (first polymorphism) selected from the group consisting of the variations set out in Table 23 (eg, selected from the group consisting of rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982), wherein the antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide sequence comprising said selected SNP.

A method of treating cancer in a human, the method comprising administering an antibody or antibody fragment to the human, wherein the antibody or antibody fragment inhibits the binding of PD-L1 or PD-L2 to a PD-1 comprising a first amino acid polymorphism, wherein the antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide sequence encoding a PD-1 comprising said first amino acid polymorphism or the human expresses a PD-1 comprising said first amino acid polymorphism.

In an alternative, instead of “A method of cancer immunotherapy” or “A method of treating cancer”, the invention instead provides “A method of treating or reducing the risk of an autoimmune disease or condition” or “A method of treating or reducing the risk inflammatory disease or condition” and the TOI polymorphism is associated with an autoimmune or inflammatory disease or condition.

The invention also provides a ligand (eg, a trap, an antibody or antibody fragment) for use in any of the methods.

In a Thirty-third configuration the invention provides: A method of treating or reducing the risk of a disease or condition (eg, anaemia) in a human, wherein the disease or condition is mediated by a TOI, the TOI being present in humans as a plurality of variants differing by one or more amino acid polymorphisms, the method comprising administering a ligand (eg, an antibody or antibody fragment) to the human, the ligand comprising first and second protein domains, wherein the first domain specifically binds a TOI variant comprising a first amino acid polymorphism, wherein the second domain comprises a second polymorphism, and wherein the human expresses (i) TOI comprising said first amino acid polymorphism; and (ii) protein domains comprising said second polymorphism, optionally wherein the ligand comprises a human gamma-4 heavy chain constant region that comprises a Len at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein the second domain is comprised by said gamma-4 heavy chain constant region of the ligand; wherein, the TOI is selected from the human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis.

Examples of any configuration or relating to any TOI are as follows:—

(i) wherein the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23*04), a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40. (ii) wherein the ligand comprises a VH domain derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment, and wherein said human comprises a IGHV3-7*01 VH gene segment or the human expresses VH domains derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment. (iii) wherein the ligand comprises a Vκ domain derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment, and wherein said human comprises a IGKV1-12*01 Vκ gene segment or the human expresses Vκ domains derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment. (iv) wherein the ligand comprises a Vκ domain derived from the recombination of a human Vκ segment and a human Jκ segment, the human Vκ segment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36 and wherein said human comprises a Vκ gene segment encoding a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the human expresses Vκ domains that comprise a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 and wherein said human comprises a Vκ gene segment encoding a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 or the human expresses Vκ domains that comprise a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38. (v) wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4. (vi) wherein the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6. (vii) wherein the ligand comprises a human kappa chain constant region that comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16 and wherein said human comprises (i) an IGKC1*01 human kappa chain constant region gene segment, or the human expresses antibodies comprising human kappa chain constant regions comprising a Val corresponding to position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16. (viii) wherein the ligand comprises a human IGLC1*01 lambda chain constant region and wherein said human comprises (i) a human IGLC1*01 lambda chain constant region gene segment, or the human expresses antibodies comprising human IGLC1*01 lambda chain constant regions. (ix) wherein the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73. (x) wherein the ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3*01) constant region gene segment and wherein said human comprises (i) said first constant region gene segment (eg, an IGHG3*01), or the human expresses antibodies comprising human gamma-3 heavy chain constant regions encoded by said first human IGHG3 (eg, IGHG3*01) constant region gene segment. (xi) wherein the ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human epsilon heavy chain constant regions encoded by said first constant region gene segment. (xii) wherein the ligand comprises a human mu heavy chain constant region encoded by a first human mu heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human mu heavy chain constant regions encoded by said first constant region gene segment. (xiii) wherein the ligand comprises a human alpha heavy chain constant region encoded by a first human alpha heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human alpha heavy chain constant regions encoded by said first constant region gene segment. (xiv) wherein the ligand comprises a human delta heavy chain constant region encoded by a first human delta heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human delta heavy chain constant regions encoded by said first constant region gene segment. (xv) wherein the ligand comprises a human kappa light chain constant region encoded by a first human kappa light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human kappa light chain constant regions encoded by said first constant region gene segment. (xvi) wherein the ligand comprises a human lambda light chain constant region encoded by a first human lambda light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human lambda light chain constant regions encoded by said first constant region gene segment.

In some embodiments, said ligand (eg, antibody or antibody fragment) is administered by inhaled, intravenous or subcutaneous administration and/or is comprised in an inhalable or injectable preparation.

In some embodiments, the human gamma-4 heavy chain constant region of the ligand comprises the amino acid sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof.

In some embodiments, the human gamma-4 heavy chain constant region comprises 228P and 235E.

Optionally in the present invention the TOI is selected from the group consisting of human TOIs: PCSK9, IL6R, IL4Ra, VEGF-A, Placental growth factor (PGF), PDGF-B, PDGFR-B, Ang-2, Nav1.7, Nav1.8, Nav1.9, PD-1, PD-L1, ICOS, BMP6, hemojuvelin, ferroportin, TMPRSS6, transferrin, human hemochromatosis protein (HFE) and sclerostin.

For example, the TOI is PCSK9.

For example, the TOI is IL6R.

For example, the TOI is IL4Ra.

For example, the TOI is VEGF-A.

For example, the TOI is Placental growth factor (PGF).

For example, the TOI is PDGF-B.

For example, the TOI is PDGFR-B.

For example, the TOI is Ang-2.

For example, the TOI is Nav1.7.

For example, the TOI is Nav1.8.

For example, the TOI is Nav1.9.

For example, the TOI is PD-1.

For example, the TOI is PD-L1.

For example, the TOI is ICOS.

For example, the TOI is BMP6.

For example, the TOI is hemojuvelin.

For example, the TOI is ferroportin.

For example, the TOI is TMPRSS6.

For example, the TOI is transferrin.

For example, the TOI is human hemochromatosis protein (HFE).

For example, the TOI is sclerostin.

In a Thirty-fourth configuration, described herein is a method of treating or reducing the risk of a Nav1.8-mediated disease or condition (eg, a pain or cardiac condition) in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain); wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said selected amino acid. The invention also provides a corresponding ligand, eg, antibody or antibody fragment, for use in such a method.

In alternative embodiments, a VH or VL domain of the ligand (eg, antibody) and/or a different constant region is matched according to the invention.

In a Thirty-fifth configuration, described herein is a method of treating or reducing the risk of a Nav1.9-mediated disease or condition (eg, a pain condition) in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E; wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said selected amino acid. The invention also provides a corresponding ligand, eg, antibody or antibody fragment, for use in such a method.

In alternative embodiments, a VH or VL domain of the ligand (eg, antibody) and/or a different constant region is matched according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows in silico modeling of PCSK9 surface variant residues.

FIG. 2 depicts the cumulative allele frequency distribution across the 1000 Genomes Project database of human VH3-23 alleles comprising SNP rs56069819 (such alleles denoted “C” and the most frequent allele (which does not comprise this SNP) denoted “A”). The figure shows that VH3-23 alleles comprising SNP rs56069819 are present at a cumulative frequency of 11% across all human ethnic populations taken as a whole, whereas in certain specific human ethnic sub-populations (ASW, LWK, YRI, CEU and GBR) such alleles are present at an above-average cumulative frequency. Indicated in the figure are those human PCSK9 variant forms (marked “Variants”) that are found in the various sub-populations with above-average occurrence of human VH3-23 alleles comprising SNP rs56069819.

FIG. 3 depicts frameworks and CDRs encoded by VH3-23*04 as obtained from the IMGT database (available on the World Wide Web at www.IMGT.org). FIG. 3 discloses the nucleotide sequences as SEQ ID NOS 117, 117, 117, 119, 119, 119, 120, 122, 124, 39 and 125, respectively, in order of appearance. FIG. 3 discloses the coded amino acid sequences as SEQ ID NOS 118, 118, 118, 118, 118, 118, 121, 123, 123, 38 and 126, respectively, in order of appearance.

FIG. 4 depicts sequences of VH3-23*04. The portion of VH3-23*04 comprising the FW1 residue change of rs56069819 (SEQ ID NO: 38). The portion of the nucleic acid sequence encoding rs56069819 is depicted (SEQ ID NO: 39). The FW1 encoded by VH3-23*04 is depicted (SEQ ID NO: 40).

DETAILED DESCRIPTION

The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in conformation or activity of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to tailor medicines and diagnosis of patients more effectively. The present invention provides for tailored pharmaceuticals and testing that specifically addresses rarer variant forms of a human target of interest (TOI).

The present invention harnesses the power of human genetic variation analysis and rationally-designed sequence selection. The technical applications of these approaches, as per the present invention, contribute to better treatment, prophylaxis and diagnosis in humans and provides for patient benefit by providing choice and enabling personalized medicines and therapies. This provides advantages of better prescribing, less wastage of medications and improved chances of drug efficacy and better diagnosis in patients.

As sources of genomic sequence variation data, the skilled person will be aware of the available databases and resources (including updates thereof) provided by the following:—

-   1. HapMap (The International HapMap Consortium. 2003; available on     the World Wide Web at hapmap.ncbi.nlm.nih.gov/index.html.en). The     HapMap Project is an international project that aims to compare the     genetic sequences of different individuals to identify chromosomal     regions containing shared genetic variants. The HapMap www site     provides tools to identify chromosomal regions and the variant     therein, with options to drill down to population level frequency     data. -   2. 1000 Genomes Project (The 1000 Genomes Project Consortium 2010;     available on the World Wide Web at 1000genomes.org/). This resource     provides complete genomic sequence for at least 2500 unidentified     individuals from one of 25 distinct population groups. -   3. Japanese SNP Database (H. Haga et al. 2002; available on the     World Wide Web at snp.ims.u-tokyo.ac.jp/index.html). Based on a     study identifying 190,562 human genetic variants.

The present invention involves the identification and cataloguing of naturally-occurring human genomic target sequence variants, including those found to be relatively low-frequency or rare variants that segregate with specific human ethnic populations and in many individual humans.

An aspect of the invention is based on rational design of sequence selection addressing the desirability to tailor medicaments and diagnostics to rarer, but yet still significant groups of human individuals that suffer from, or have the potential to suffer from (ie, who are at risk of), a disease or condition mediated or associated with the target of interest. In devising this rational design of the present aspect of the invention, the inventor included considerations of the spread of prevalence of naturally-occurring target variant sequences across multiple, diverse human ethnic populations, as well as the importance of addressing such populations where many individuals are likely to display a genotype and/or phenotype of one or more of the variants being analysed. As part of this design, the inventor saw the importance of adopting the art-recognised classifications of human ethnic populations, and in this respect the inventor based the analysis and design on the recognised human ethnic populations adopted by the 1000 Genomes Project, since this is a resource that is, and will continue to be, widely adopted by the scientific and medical community.

Thus, in this aspect of the invention, the inventor designed the following variant sequence selection criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population.

Selection Criteria

Three or four of the following:—

-   -   Naturally-occurring human target variant sequences having a         cumulative human allele frequency of 35% or less;     -   Naturally-occurring human target variant sequences having a         total human genotype frequency of 40% or less;     -   Naturally-occurring human target variant sequences found in many         different human ethnic populations (using the standard         categorisation of the 1000 Genomes Project; see Table 4 below);         and     -   Naturally-occurring human target variant sequences found in many         individuals distributed across such many different ethnic         populations.

The inventor's selection included, as a consideration, selection for nucleotide variation that produced amino acid variation in corresponding TOI forms (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.

In an embodiment, the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to 10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to 15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about 5%.

In an embodiment, the naturally-occurring human target variant sequences are found in at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different human ethnic populations (using the standard categorisation of the 1000 Genomes Project).

In an embodiment, the naturally-occurring human target variant sequences are found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 individuals distributed across such many different ethnic populations.

In an example, the following criteria are applied:—

-   -   Naturally-occurring human target variant sequences having a         cumulative human allele frequency of 15% or less;     -   Naturally-occurring human target variant sequences having a         total human genotype frequency of 20% or less;     -   Naturally-occurring human target variant sequences found in at         least 5 different human ethnic populations (using the standard         categorisation of the 1000 Genomes Project); and     -   Naturally-occurring human target variant sequences found in many         individuals distributed across such many different ethnic         populations.

In an example, the criteria are applied with reference to one or more human genomic sequence databases as described herein. For example, the criteria are those as applied to the 1000 Genomes database.

For example in any aspect example, embodiment or configuration of the invention, the 1000 Genomes database release 13. For example, the 1000 Genomes database in its most recent version as at 1 Oct. 2013.

Optionally, further sequence analysis and 3D in silico modelling (eg, see FIG. 1 ) can also be used as an additional selection criterion: variants whose variant amino acid residues (versus the most common form of human TOI) are surface-exposed on the target are desirable for selection, since the inventor saw these as contributing to determining the topography of the target and potentially contributing to how and where ligand binding on the target occurs.

The following bioinformatics protocol is envisaged to indentify human sequences for use in the present invention:

-   -   (a) Identify a genomic region containing a target sequence of         interest (‘target genomic region’) and calculate the genomic         coordinates, using coordinates that match the sequence assembly         build used by either the 1000 Genomes Project or International         HapMap project (or another selected human gene database of         choice).     -   (b) Identify genomic variants mapped to the genomic region         previously identified in (a). Retrieve allele frequencies for         variants for each super population and preferably sub-population         where such data is available. The VWC tools for the 1000 Genomes         Project can be used for this step.     -   (c) Filter list of genomic variants from target genomic region         to contain only variants classed as either ‘non-synonymous’         single nucleotide polymorphisms (SNPs) or genomic ‘insertions or         delections’ (indels). Filter further to include those that are         present in exonic sequences only. “Non-synonymous” refers to         nucleotide variation that produces amino acid variation (ie,         excluding silent mutations).     -   (d) Correlate population frequency data for each of the         identified variants for each of the super populations (for         example ‘European Ancestry’, ‘East Asian ancestry’, ‘West         African ancestry’, ‘Americas’, and ‘South Asian ancestry’) to         identify those variants that segregate with less than two         super-populations. Further correlate all identified variants         with each of the sub-populations (for example, ‘European         ancestry’ super-population might be subdivided into groups such         as ‘CEU—Utah residents with Northern or Western European         ancestry’, ‘TSI Toscani in Italia’ and ‘British from England and         Scotland’) and produce a second score for rarity of variants         within a super-population.     -   (e) Collect one or more sequences that show segregation to         specific sub-populations for use in the present invention, eg,         according to selection criteria as described herein.

Human Populations

Optionally the ethnic populations are selected from those identified in the 1000 Genomes Project database. In this respect, see Table 4 which provides details of the ethnic populations on which the 1000 Genomes Project database is based.

N A Rosenberg et al (Science 20 Dec. 2002: vol. 298 no. 5602 2342-2343) studied the genetic structure of human populations of differing geographical ancestry. In total, 52 populations were sampled, these being populations with:

African Ancestry

(Mbuti Pygmies, Biaka Pygmies, San peoples, and speakers of Niger-Kordofanian languages (Bantu, Yoruba or Mandenka populations),

Eurasian Ancestry

(European ancestry (Orcadian, Adygel, Basque, French, Russians, Italians, Sardinian, Tuscan),

Middle Eastern ancestry (Mozabite, Bedouin, Druze, Palestinians),

Central/South Asian ancestry (Balochl, Brahul, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash)),

East Asian Ancestry

(Han, Dal, Daur, Hezhen, Lahu, Miao, Orogen, She, Tujia, Tu, Xibo, Yi, Mongola, Naxi, Cambodian, Japanese, Yakut), Oceanic ancestry (Melanesian, Papuan); or

Americas Ancestry

(Karitiana, Surui, Colombian, Maya, Pima).

The International HapMap Project, Nature, 2003 Dec. 18; 426(6968):789-96, discloses that goal of the HapMap Project: to determine the common patterns of DNA sequence variation in the human genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The relevant human populations of differing geographical ancestry include Yoruba, Japanese, Chinese, Northern European and Western European populations. More specifically:—

Utah population with Northern or Western European ancestry (samples collected in 1980 by the Centre d'Etude du Polymorphisme Humain (CEPH));

population with ancestry of Yoruba people from Ibadan, Nigeria; population with Japanese ancestry; and population with ancestry of Han Chinese from China.

The authors, citing earlier publications, suggest that ancestral geography is a reasonable basis for sampling human populations.

A suitable sample of human populations used in the present invention is as follows:—

-   -   (a) European ancestry     -   (b) Northern European ancestry; Western European ancestry;         Toscani ancestry; British ancestry, Finnish ancestry or Iberian         ancestry.     -   (c) More specifically, population of Utah residents with         Northern and/or Western European ancestry; Toscani population in         Italia; British population in England and/or Scotland; Finnish         population in Finland; or Iberian population in Spain.     -   (a) East Asian ancestry     -   (b) Japanese ancestry; Chinese ancestry or Vietnamese ancestry.     -   (c) More specifically, Japanese population in Toyko, Japan; Han         Chinese population in Beijing, China; Chinese Dai population in         Xishuangbanna; Kinh population in Ho Chi Minh City, Vietnam; or         Chinese population in Denver, Colo., USA.     -   (a) West African ancestry     -   (b) Yoruba ancestry; Luhya ancestry; Gambian ancestry; or         Malawian ancestry.     -   (c) More specifically, Yoruba population in Ibadan, Nigeria;         Luhya population in Webuye, Kenya; Gambian population in Western         Division, The Gambia; or Malawian population in Blantyre,         Malawi.     -   (a) Population of The Americas     -   (b) Native American ancestry; Afro-Caribbean ancestry; Mexican         ancestry; Puerto Rican ancestry; Columbian ancestry; or Peruvian         ancestry.     -   (c) More specifically, population of African Ancestry in         Southwest US; population of African American in Jackson, M S;         population of African Caribbean in Barbados; population of         Mexican Ancestry in Los Angeles, Calif.; population of Puerto         Rican in Puerto Rico; population of Colombian in Medellin,         Colombia; or population of Peruvian in Lima, Peru.     -   (a) South Asian ancestry     -   (b) Ahom ancestry; Kayadtha ancestry; Reddy ancestry; Maratha;         or Punjabi ancestry.     -   (c) More specifically, Ahom population in the State of Assam,         India; Kayadtha population in Calcutta, India; Reddy population         in Hyderabad, India; Maratha population in Bombay, India; or         Punjabi population in Lahore, Pakistan.

In any configuration of the invention, in one embodiment, each human population is selected from a population marked “(a)” above.

In any configuration of the invention, in another embodiment, each human population is selected from a population marked “(b)” above.

In any configuration of the invention, in another embodiment, each human population is selected from a population marked “(c)” above.

In one embodiment the ethnic populations are selected from the group consisting of an ethnic population with European ancestry, an ethnic population with East Asian, an ethnic population with West African ancestry, an ethnic population with Americas ancestry and an ethnic population with South Asian ancestry.

In one embodiment the ethnic populations are selected from the group consisting of an ethnic population with Northern European ancestry; or an ethnic population with Western European ancestry; or an ethnic population with Toscani ancestry; or an ethnic population with British ancestry; or an ethnic population with Icelandic ancestry; or an ethnic population with Finnish ancestry; or an ethnic population with Iberian ancestry; or an ethnic population with Japanese ancestry; or an ethnic population with Chinese ancestry; or an ethnic population Vietnamese ancestry; or an ethnic population with Yoruba ancestry; or an ethnic population with Luhya ancestry; or an ethnic population with Gambian ancestry; or an ethnic population with Malawian ancestry; or an ethnic population with Native American ancestry; or an ethnic population with Afro-Caribbean ancestry; or an ethnic population with Mexican ancestry; or an ethnic population with Puerto Rican ancestry; or an ethnic population with Columbian ancestry; or an ethnic population with Peruvian ancestry; or an ethnic population with Ahom ancestry; or an ethnic population with Kayadtha ancestry; or an ethnic population with Reddy ancestry; or an ethnic population with Maratha; or an ethnic population with Punjabi ancestry.

Anti-Target Ligands

The invention provides useful anti-target ligands for addressing humans suffering from or likely to suffer from a disease or condition mediated or associated with the TOI. For example, the ligand specifically binds to the TOI variant as per the invention. The ligand may inhibit or antagonise the activity of the target, eg, the ligand neutralises the target. The skilled person will be familiar with neutralising ligands in general, such as antibodies or antibody fragments, and can readily test suitable ligands for specific binding and/or neutralisation of a target in vitro or in an in vivo assay.

An antibody “fragment” comprises a portion intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include dAb, Fab, Fab′, F(ab′)2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

In an embodiment, the ligand of the invention is or comprises an antibody or antibody fragment, for example an antibody or fragment comprising human variable regions (and optionally also human constant regions). Anti-TOI or TOI-binding or targeting antibodies and fragments can be prepared according to any known method, eg, using transgenic mice (eg, the Kymouse™ or Velocimouse™, or Omnimouse™, Xenomouse™, HuMab Mouse™ or MeMo Mouse™), rats (eg, the Omnirat™), camelids, sharks, rabbits, chickens or other non-human animals immunised with the TOI followed optionally by humanisation of the constant regions and/or variable regions to produce human or humanised antibodies. In an example, display technologies can be used, such as yeast, phage or ribosome display, as will be apparent to the skilled person. Standard affinity maturation, eg, using a display technology, can be performed in a further step after isolation of an antibody lead from a transgenic animal, phage display library or other library. Representative examples of suitable technologies are described in US20120093818 (Amgen, Inc), which is incorporated herein by reference, eg, the methods set out in paragraphs [0309] to [0346]. Although this is with reference to PCSK9, the antibody-generating methods can be applied to other TOIs as per the broadest scopes of the present invention.

Generally, a VELOCIMMUNE™ or other mouse or rat can be challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimaeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

Initially, high affinity chimaeric antibodies are isolated having a human variable region and a mouse constant region. As described below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4 (for example, SEQ ID NO: 751, 752, 753 in US2011/0065902, which sequences are incorporated herein by reference for use in the ligands of the present invention). While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

In an example, the ligand of the invention is or comprises a nucleic acid, eg, RNA, eg, siRNA that hybridises under stringent condition to the TOI variant sequence, eg, hybridises a nucleotide sequence comprising one or more nucleotides that are variant (versus the most common TOI sequence, eg, with reference to the 1000 Genomes Project database).

Target binding ability, specificity and affinity (Kd, K_(off) and/or K_(on)) can be determined by any routine method in the art, eg, by surface plasmon resonance (SPR). The term “Kd”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

In one embodiment, the surface plasmon resonance (SPR) is carried out at 25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such as about pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (also referred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level, eg, 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of no greater than 0.05% by volume, eg, in the presence of P20 (polysorbate 20; eg, Tween-20′) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a buffer at pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. The buffer can contain 10 mM Hepes. In one example, the SPR is carried out at 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc (California; catalogue number H8022).

In an example, the affinity of the ligand (eg, antibody) is determined using SPR by

-   1. Coupling anti-mouse (or other relevant human, rat or non-human     vertebrate antibody constant region species-matched) IgG (eg,     Biacore™ BR-1008-38) to a biosensor chip (eg, GLM chip) such as by     primary amine coupling; -   2. Exposing the anti-mouse IgG (or other matched species antibody)     to a test IgG antibody to capture test antibody on the chip; -   3. Passing the test antigen over the chip's capture surface at 1024     nM, 256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and -   4. And determining the affinity of binding of test antibody to test     antigen using surface plasmon resonance, eg, under an SPR condition     discussed above (eg, at 25° C. in physiological buffer). SPR can be     carried out using any standard SPR apparatus, such as by Biacore™ or     using the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mM glycine at pH1.7. This removes the captured antibody and allows the surface to be used for another interaction. The binding data can be fitted to 1:1 model inherent using standard techniques, eg, using a model inherent to the ProteOn XPR36™ analysis software.

In an example, the ligand of the invention is contained in a medical container, eg, a vial, syringe, IV container or an injection device (eg, an intraocular or intravitreal injection device). In an example, the ligand is in vitro, eg, in a sterile container. In an example, the invention provides a kit comprising the ligand of the invention, packaging and instructions for use in treating or preventing or diagnosing in a human a disease or condition mediated by the TOI. In an example, the instructions indicate that the human should be genotyped for a TOI variant sequence of the invention before administering the ligand to the human. In an example, the instructions indicate that the human should be phenotyped for a TOI variant of the invention before administering the ligand to the human. In an example, the human is of Chinese (eg, Han) ethnicity and the instructions are in Chinese (eg, Mandarin). In an example, the instructions comprise directions to administer alirocumab or evolocumab to said human.

The invention relates to the concepts set out in the following clauses.

Clause 1 A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI         variant in the human and treat or prevent (eg, by at least 40,         50, 60, 70, 80, 90 or 95%) said disease or condition, wherein         the TOI in said human is encoded by a nucleotide sequence having         a cumulative human allele frequency of less than 50% and/or         wherein the TOI in said human is encoded by a nucleotide         sequence having a total human genotype frequency of less than         50%;         -   wherein         -   Before step (a) said human has been or is genotyped as             positive for said nucleotide sequence or phenotyped as             positive for said TOI variant, or the method comprises             before step (a) genotyping the human as positive for said             nucleotide sequence or phenotyping the human as positive for             said TOI variant.

In any aspect, configuration, example, embodiment, clause or concept herein, frequencies may be determined using bioinformatics.

In any aspect, configuration, example, embodiment, clause or concept herein, frequencies may be determined by reference to a database comprising at least 1000 or 2000 human sequences.

In any aspect, configuration, example, embodiment, clause or concept herein “heterozygous human genotype frequency” means the cumulative frequency of all genotypes in the sample or database or in humans having one occurrence of the rare variant allele and one occurrence of another allele (heterozygous state), eg, genotype in 1000 Genomes database.

In any aspect, configuration, example, embodiment, clause or concept herein “homozygous human genotype frequency means the cumulative frequency of two occurrences of the variant allele (homozygous state), eg, genotype in 1000 Genomes Project database.

In any aspect, configuration, example, embodiment, clause or concept herein “total human genotype frequency” means the total of heterozygous plus homozygous human genotype frequencies.

In any aspect, configuration, example, embodiment, clause or concept herein “cumulative human allele frequency” refers to the total of all occurrences of the variant allele (eg, SNP) in the sample or database or in humans, eg, in the 1000 Genomes Project database.

Clause 2: The method of clause 1, wherein before step (a) the ligand has been or is determined as being capable of binding to said TOI variant, eg, with an affinity (Kd) disclosed below.

In an example, the ligand is (or has been determined as) a neutraliser of the TOI. In an example, determination is carried out in a human (eg, in a clinical trial). In an example, determination is carried out in a non-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-human primate (eg, Cynomolgous monkey, rhesus monkey or baboon).

Clause 3: A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI         variant in the human and treat or prevent (eg, by at least 40,         50, 60, 70, 80, 90 or 95%) said disease or condition, wherein         the TOI in said human is encoded by a nucleotide sequence having         a cumulative human allele frequency of less than 50% and/or         wherein the TOI in said human is encoded by a nucleotide         sequence having a total human genotype frequency of less than         50%;         -   wherein     -   b. Before step (a) the ligand has been or is determined as         capable of binding to said TOI variant, eg, with an affinity         (Kd) disclosed below.

In an example, the ligand is (or has been determined as) a neutraliser of the TOI. In an example, determination is carried out in a human (eg, in a clinical trial). In an example, determination is carried out in a non-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-human primate (eg, Cynomolgous monkey, rhesus monkey or baboon).

Clause 4: The method of clause 3, wherein the genome of said human comprises a nucleotide sequence encoding said TOI variant; and before step (a) said nucleotide sequence has been or is determined as having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

The TOI variant is not the most frequent.

Clause 5: The method of clause 3 or 4, wherein said human has been or is genotyped as positive for said variant nucleotide sequence before step (a), or the method comprises genotyping the human as positive for said variant nucleotide sequence before step (a).

Clause 6: The method of any preceding clause, wherein the human has been or is phenotyped as positive for said TOI variant before step (a), or the method comprises phenotyping the human as positive for said variant nucleotide sequence before step (a).

Clause 7: The method of any preceding clause, wherein said frequency is less than 10 or 15% (eg, from 1 to 10%).

In an embodiment, the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to 10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to 15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about 5%.

Clause 8: The method of any preceding clause, wherein the ligand is capable of binding to two or more different TOI variants, each being encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% (eg, from 1 to 10%) and/or having a total human genotype frequency of less than 50% (eg, from 1 to 20%).

In an embodiment, the cumulative human allele frequency of each TOI variant is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to 10%.

In an embodiment, the total human genotype frequency of each TOI variant is 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to 15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about 5%.

Clause 9: A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI         variant in the human and treat or prevent (eg, by at least 40,         50, 60, 70, 80, 90 or 95%) said disease or condition, wherein         the TOI in said human is a variant encoded by a nucleotide         sequence having a cumulative human allele frequency of more than         50% (eg, the highest frequency) and/or having a total human         genotype frequency of more than 50% (eg, the highest frequency);         -   wherein     -   b. Before step (a) said human has been or is genotyped as         negative for a variant nucleotide sequence having a cumulative         human allele frequency of less than 50% and/or having a total         human genotype frequency of less than 50%; or phenotyped as         negative for a TOI variant encoded by a nucleotide sequence         having a cumulative human allele frequency of less than 50%         and/or having a total human genotype frequency of less than 50%;         -   or         -   Before step (a) said the method comprises genotyping the             human as negative for a variant nucleotide sequence having a             cumulative human allele frequency of less than 50% and/or             having a total human genotype frequency of less than 50%; or             phenotyping the human as negative for a TOI variant encoded             by a nucleotide sequence having a cumulative human allele             frequency of less than 50% and/or having a total human             genotype frequency of less than 50%.

In an embodiment, in (a) the cumulative human allele frequency is 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%).

In an embodiment, in (a) the total human genotype frequency is 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%).

In an embodiment, in (b) the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to 10%.

In an embodiment, in (b) the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to 15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about 5%.

Clause 10: The method of clause 9, wherein before step (a), the human has been or is phenotyped as positive for the most frequent TOI variant or genotyped for the nucleotide sequence thereof.

In an embodiment, before step (a) the human has been or is genotyped as positive for TOI variant nucleotide sequence having a cumulative human allele frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%) or phenotyped for the TOI variant thereof.

In an embodiment, before step (a) the human has been or is genotyped as positive for TOI variant nucleotide sequence having a total human genotype frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%) or phenotyped for the TOI variant thereof.

Clause 11: The method of clause 9 or 10, wherein before step (a) the ligand has been or is determined as being capable of binding to the most frequent TOI variant.

Clause 12: The method of clause 9, 10 or 11, wherein before step (a) the ligand has been or is determined as being substantially incapable of neutralising or inhibiting said TOI variant recited in step (b).

By “substantially incapable or neutralising or inhibiting” is meant: Neutralisation or inhibition less than 50, 25, 10, 5 or 0.5% inhibition or neutralisation of the most frequent TOI variant.

Clause 13: The method of any one of clauses 9 to 12, wherein the ligand is capable of binding to the most frequent TOI variant.

Clause 14: The method of any one of clauses 9 to 13, wherein the ligand is capable of binding to two or more different TOI variants, each being encoded by a nucleotide sequence having a cumulative human allele frequency of more than 50%.

In an embodiment, each TOI variant is encoded by a nucleotide sequence having a cumulative human allele frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%).

In an embodiment, each TOI variant is encoded by a nucleotide sequence having a total human genotype frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%).

Clause 15: The method of any preceding clause, wherein said variant nucleotide sequence recited in step (a) has been or is determined as being present in at least 2 different human ethnic populations, eg, at least 2, 3, 4, 5, 6, 7, 8 or 9 different human ethnic populations in Table 4.

Clause 16: The method of any preceding clause, wherein said human frequency is the frequency in a database of naturally-occurring sequences sampled from at least 15, 20 or 25 different human ethnic populations and comprising at least 1000 sequences. In an embodiment, the database is the 1000 Genomes Project database as described herein.

Clause 17: An anti-human TOI ligand for use in a method of treating and/or preventing a TOI-mediated disease or condition in a human, wherein the TOI is present in humans as different polymorphic variants and wherein the genome of said human comprises a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, the method comprising administering the ligand to the human.

In the alternative, clause 17 provides an anti-human TOI ligand for use in a method according to any one of clauses 1 to 16, the method comprising administering the ligand to the human.

In an embodiment, the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to 10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to 15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about 5%.

Clause 18: The ligand of clause 17, wherein the ligand has been or is determined as capable of binding the human TOI encoded by said nucleotide sequence.

In the alternative, clause 18 provides a ligand that binds a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, for use in a method comprising the step of using the ligand to target said TOI in a human to treat and/or prevent a disease or condition mediated by TOI, the method comprising administering the ligand to the human.

Clause 19: A ligand that binds a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, for use in a method according to any one of clauses 1 to 16, the method comprising administering the ligand to the human.

Clause 20: The ligand of any one of clauses 17 to 19, wherein the human has been or is genotyped as positive for said TOI nucleotide sequence having a cumulative human allele frequency of less than 50%.

The ligand of any one of clauses 17 to 19, wherein the human has been or is genotyped as positive for said TOI nucleotide sequence having a total human genotype frequency of less than 50%.

Clause 21: The ligand of any one of clauses 17 to 20, wherein the human has been or is phenotyped as positive for a TOI encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

Clause 22: The ligand of any one of clauses 17 to 21, wherein the human has been or is genotyped as heterozygous for a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; optionally wherein the human has been or is genotyped as comprising a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and a TOI nucleotide sequence having a cumulative human allele frequency of more than 50% (eg, having the highest cumulative human allele frequency) and/or having a total human genotype frequency of more than 50% (eg, having the highest total human genotype frequency).

Clause 23: The ligand of any one of clauses 17 to 22, wherein the genome of the human has been or is genotyped as homozygous for a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

Clause 24: The ligand of any one of clauses 17 to 23, wherein the ligand comprises an antibody binding site that binds a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; and optionally has been or is determined as capable of such binding.

Clause 25: The ligand of clause 24, wherein the ligand is an antibody or antibody fragment.

Clause 26:The ligand of any one of clauses 17 to 23, wherein the ligand comprises a nucleotide sequence that specifically hybridises to a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or an RNA transcript thereof; and/or the ligand comprises a nucleotide sequence that comprises at least 10 contiguous nucleotides of a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or is an antisense sequence thereof.

In an embodiment, the ligand comprises a nucleotide sequence that comprises at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or 100 contiguous nucleotides of a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or is an antisense sequence thereof.

Clause 27: The ligand of any one of clauses 17 to 26, wherein the genome of said human comprises a nucleotide sequence having a cumulative human allele frequency of less than 50% and the sequence is found in at least 2 different ethnic populations (eg, found in at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different human ethnic populations (for example as per the populations in Table 4)). In an example, numbers are with reference to the 1000 Genomes Project database.

The ligand of any one of clauses 17 to 26, wherein the genome of said human comprises a nucleotide sequence having a cumulative human allele frequency of less than 50% and the sequence is found in at least 20 individuals distributed across at least 2 said different ethnic populations (eg, found in at least in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 individuals distributed across such many different ethnic populations). In an example, numbers are with reference to the 1000 Genomes Project database.

Clause 28: A pharmaceutical composition or kit for treating or preventing a condition or disease mediated by a TOI as recited in any preceding clause, the composition or kit comprising a ligand of any one of clauses 17 to 27; and optionally in combination with a label or instructions for use to treat and/or prevent said disease or condition in a human; optionally wherein the label or instructions comprise a marketing authorisation number (eg, an FDA or EMA authorisation number); optionally wherein the kit comprises an injection pen or IV container that comprises the ligand.

In an example, the label or instructions cover or describe use for a human comprising a TOI variant encoded by a nucleotide sequence as recited in clause 17.

Clause 29: A method of producing an anti-human TOI antibody binding site, the method comprising obtaining a plurality of anti-TOI antibody binding sites, screening the antibody binding sites for binding to a TOI comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOI-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody binding site that binds in the screening step.

In an embodiment of any aspect herein, the antibody, fragment or binding site is recombinant.

In the alternative, clause 29 provides: A method of producing an anti-human TOI antibody, the method comprising immunising a non-human vertebrate (eg, a mouse or a rat) with a TOI comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOI-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody that binds a TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, and optionally producing a TOI-binding fragment or derivative of the isolated antibody.

The term “isolated” with reference to a ligand, antibody or protein, for example in any aspect, configuration, example or embodiment, means that a subject ligand, antibody, protein etc (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature. Typically, an “isolated” ligand, antibody, protein etc constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode such an isolated ligand, antibody protein etc. Preferably, the isolated ligand, antibody protein etc is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.

For example, an “isolated” antibody is one that has been identified, separated and/or recovered from a component of its production environment (eg, naturally or recombinantly). Preferably, the isolated polypeptide is free of association with all other components from its production environment, eg, so that the antibody has been isolated to an FDA-approvable or approved standard. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.

Immunoconjugates

The invention encompasses the ligand (eg, antibody) conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxin agents include any agent that is detrimental to cells. Examples of suitable cytotoxin agents and chemotherapeutic agents for forming immunoconjugates are known in the art, see for example, WO 05/103081.

Bispecifics

The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific mAbs may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al. (1991) J. Immunol. 147:60-69. The human anti-TOI (eg, anti-PCSK9) mAbs can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody with a second binding specificity.

An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N3845, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.

Clause 30: The method of clause 29, comprising the step of obtaining a nucleic acid encoding the antibody, fragment, derivative or binding site and optionally inserting the nucleic acid in an expression vector.

Clause 31: A kit for TOI genotyping a human, wherein the kit comprises a nucleic acid comprising a nucleotide sequence that specifically hybridises to a TOI nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or an RNA transcript thereof; and/or the nucleic acid comprises a nucleotide sequence that comprises at least 10 (eg, at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or 100) contiguous nucleotides of a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or is an antisense sequence thereof.

For example, the nucleic acid hybridises to a region immediately flanking a nucleotide that is variant compared to the corresponding nucleotide of the TOI nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency. In an example, the nucleic acid hybridises to at two or more such variant nucleotides.

Specific hybridisation is under stringent conditions, as will be apparent to the skilled person, eg, conditions of 5×SSC, 5×Denhardt's reagent, and 0.5% SDS at 65° C.

Clause 32: A kit for TOI genotyping or phenotyping a human, wherein the kit comprises a ligand according to any one of clauses 17 to 27 or an antibody, fragment or derivative produced by the method of any one of clauses 29 to 31.

For example, the ligand specifically binds to an epitope comprising an amino acid that is variant compared to the corresponding amino acid of the TOI encoded by a nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency. In an example, the ligand specifically binds to an epitope comprising two or more such variant amino acids. In an example, specific binding means binding with an affinity (Kd) of 1 mM, 100 nM, 10 nM or 1 nM or less, eg, as determined by SPR.

The term “epitope” is a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.

Clause 33: Use of an anti-TOI ligand that binds a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for treating and/or preventing a TOI-mediated disease or condition in a human whose genome comprises a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

Clause 34: Use of an anti-TOI ligand that binds a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for targeting said TOI in a human to treat and/or prevent a disease or condition mediated by TOI.

The use of clause 33 or 34, wherein the ligand, human, disease or condition is according to any one of clauses 1 to 27.

Clause 35: A method of targeting a TOI for treating and/or preventing a TOI-mediated disease or condition in a human, the method comprising administering an anti-TOI ligand to a human comprising a TOI nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, whereby a TOI encoded by said nucleotide sequence is targeted.

Clause 36: The method of clause 35, wherein the method comprises targeting a human TOI comprising an amino acid sequence with said ligand to treat and/or prevent said disease or condition in said human, wherein said amino acid sequence is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%.

Clause 37: A method of TOI genotyping a nucleic acid sample of a human, the method comprising identifying in the sample the presence of a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

In an example, the method comprises obtaining a TOI nucleic acid sample from the human and then carrying out the identifying step.

Clause 38: A method of TOI typing a protein sample of a human, the method comprising identifying in the sample the presence of a TOI amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

In an example, the method comprises obtaining a TOI protein sample from the human and then carrying out the identifying step.

Clause 39: The method of clause 37 or 38, comprising obtaining a sample of serum, blood, faeces, hair, urine or saliva from a human, whereby the nucleic acid or protein sample is obtained for use in the step of identifying said sequence.

Clause 40: The method of any one of clauses 37 to 39, comprising using a ligand according to any one of clauses 17 to 27 to carry out said identifying step.

Clause 41: A diagnostic kit comprising a ligand that is capable of binding a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% and instructions for carrying out the method of clause 38 or 39.

Clause 42: A diagnostic kit comprising a nucleic acid probe comprising a nucleotide sequence that specifically hybridises a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or an RNA transcript thereof and instructions for carrying out the method of clause 38 or 39.

Clause 43: The method, ligand, composition, kit or use of any preceding clause, wherein the TOI is encoded by a nucleotide sequence having a cumulative human allele frequency from 1 to 10% and/or a total human genotype frequency from 1 to about 15% or from 1 to 15%.

Clause 44: The method, ligand, composition, kit or use of any preceding clause wherein the TOI is a human TOI selected from Table 5; optionally for treating and/or preventing a corresponding disease or condition as set out in Table 5.

For example, the TOI is human PCSK9, eg, a mature, cleaved, autocatalysed or active PCSK9. In an example, the disease is a cardiovascular disease such as hyperlipidaemia.

Ligands of the invention are useful, for instance, in specific binding assays, for genotyping or phenotyping humans, affinity purification of the TOI and in screening assays to identify other antagonists of TOI activity. Some of the ligands of the invention are useful for inhibiting binding of TOI to a congnate human receptor or protein, or inhibiting TOI-mediated activities.

The invention encompasses anti-TOI (eg, PCSK9) antibody ligands having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or e.g., removal of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

In an example, the invention features a pharmaceutical composition comprising a ligand of the invention, wherein the ligand is or comprises a recombinant human antibody or fragment thereof which specifically binds the TOI (eg, a rare variant as described herein) and a pharmaceutically acceptable carrier. In one embodiment, the invention features a composition which is a combination of an antibody ligand or antigen-binding fragment of an antibody of the invention, and a second therapeutic agent. The second therapeutic agent may be any of an anti-inflammatory agent, an anti-angiogenesis agent, a painkiller, a diuretic, a chemotherapeutic agent, an anti-neoplastic agent, a vasodilator, a vasoconstrictor, a statin, a beta blocker, a nutrient, an adjuvant, an anti-obesity agent and an anti-diabetes agent.

“Pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the USA Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. A “pharmaceutically acceptable carrier, excipient, or adjuvant” refers to an carrier, excipient, or adjuvant that can be administered to a subject, together with an agent, e.g., any antibody or antibody chain described herein, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

In an example, the invention features a method for inhibiting TOI activity using the anti-TOI ligand of the invention (eg, an antibody or antigen-binding portion of the antibody of the invention), wherein the therapeutic method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the ligand. The disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of TOI activity.

By the phrase “therapeutically effective amount” is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

The term “specifically binds,” or the like, means that a ligand, eg, an antibody or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10⁻⁶ M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. An isolated antibody that specifically binds a human TOI may, however, exhibit cross-reactivity to other antigens such as a TOI molecule from another species. Moreover, multi-specific antibodies (e.g., bispecifics) that bind to human TOI and one or more additional antigens are nonetheless considered antibodies that “specifically bind” TOI, as used herein.

Genotyping & Phenotyping

The skilled person will be familiar with techniques that can be used for accurate genotyping and application to the invention. These include the following.

1 Hybridization-based methods 1.1 Dynamic allele-specific hybridization 1.2 Molecular beacons 1.3 SNP microarrays 2 Enzyme-based methods 2.1 Restriction fragment length polymorphism 2.2 PCR-based methods 2.3 Flap endonuclease 2.4 Primer extension 2.5 5′-nuclease

2.6 Oligonucleotide Ligation Assay

3 Other post-amplification methods based on physical properties of DNA 3.1 Single strand conformation polymorphism 3.2 Temperature gradient gel electrophoresis 3.3 Denaturing high performance liquid chromatography 3.4 High-resolution melting of the entire amplicon 3.5 Use of DNA mismatch-binding proteins 3.6 SNPlex (SNPlex™ is a proprietary genotyping platform sold by Applied Biosystems).

Next-generation sequencing technologies such as pyrosequencing is also useful.

Reference is also made to GB2444410A and the genotyping method disclosed therein, which is incorporated herein by reference in its entirety.

Miniaturized assays, such as microarrays with oligonucleotide reagents immobilized on small surfaces, are frequently proposed for large-scale mutation analysis and high-throughput genotyping (Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome (Wang D G, Fan J B, Siao C J, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris M S, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson T J, Lipshutz R, Chee M, Lander E S, Science. 1998 May 15; 280(5366):1077-82). Other high-throughput methods discriminate alleles by differential hybridization, primer extension, ligation and cleavage of an allele-specific probe (Review Accessing genetic variation: genotyping single nucleotide polymorphisms, Syvanen A C, Nat Rev Genet. 2001 December; 2(12):930-42; Review Techniques patents for SNP genotyping, Twyman R M, Primrose S B, Pharmacogenomics. 2003 January; 4(1):67-79).

An approach for a fully automated, large-scale SNP analysis is the ‘homogeneous’ assay, i.e. a single-phase assay without separation steps, permitting continual monitoring during amplification. The TaqMan™ assay (Applied Biosystems), originally designed for quantitative real-time PCR, is a homogeneous, single-step assay also used in determination of mutation status of DNA (see, eg, A. A. Komar (ed.), Single Nucleotide Polymorphisms, Methods in Molecular Biology 578, DOI 10.1007/978-1-60327-411-1_19, Humana Press, a part of Springer Science+Business Media, LLC; and Single Nucleotide Polymorphisms, Methods in Molecular Biology™ Volume 578, 2009, pp 293-306, The TaqMan Method for SNP Genotyping, Gong-Qing Shen et al). The TaqMan SNP Genotyping Assay exploits the 5′-exonuclease activity of AmpliTaq Gold™ DNA polymerase to cleave a doubly labeled probe hybridized to the SNP-containing sequence of ssDNA. Cleavage separates a 5′-fluorophore from a 3′-quencher leading to detectable fluorescent signal. The use of two allele-specific probes carrying different fluorophores permits SNP determination in the same tube without any post-PCR processing. Genotype is determined from the ratio of intensities of the two fluorescent probes at the end of amplification. Thus, rather than taking advantage of the full set of real-time PCR data as in quantitative studies, only end-point data are used.

TaqMan SNP genotyping in a high-throughput, automated manner is facilitated by the use of validated Pre-made TaqMan® Genotyping assays, but Custom TaqMan® Assays may also be used (High-throughput genotyping with single nucleotide polymorphisms, Ranade K, Chang M S, Ting C T, Pei D, Hsiao C F, Olivier M, Pesich R, Hebert J, Chen Y D, Dzau V J, Curb D, Olshen R, Risch N, Cox D R, Botstein D, Genome Res. 2001 July; 11(7):1262-8; Assessment of two flexible and compatible SNP genotyping platforms: TaqMan SNP Genotyping Assays and the SNPlex Genotyping System, De la Vega F M, Lazaruk K D, Rhodes M D, Wenz M H, Mutat Res. 2005 Jun. 3; 573(1-2):111-35). The results of the assay can be automatically determined by genotyping software provided with real-time thermal cyclers (e.g. IQ software of Bio-Rad, Sequence Detection Software of Applied Biosystems).

Single nucleotide polymorphisms (SNPs) can be determined using TaqMan™ real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. An algorithm for automatic genotype caling of SNPs using the full course of TaqMan real-time data is available for use (A. Callegaro et al, Nucleic Acids Res. 2006; 34(7): e56, Published online 2006 Apr. 14. doi: 10.1093/nar/gk1185, PMCID: PMC1440877). The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare.

The skilled person will be familiar with techniques that can be used for accurate phenotyping and application to the invention. These include the use of amino acid sequencing of isolated target protein and comparison of sequences from different variants (eg, with the most common variant). An antibody that specifically and selectively binds in the area of a SNP under stringent conditions can also be used to identify a particular variant. In another method, the genotype is determined and a corresponding amino acid sequence (phenotype) determined, eg, by in silico translation.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the anti-TOI ligand, eg, antibodies or antigen-binding fragments thereof, of the present invention. The administration of therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When the ligand, eg, antibody, of the present invention is used for treating various conditions and diseases associated with the TOI in an adult patient, it is advantageous to intravenously administer the antibody of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted.

Various delivery systems are known and can be used to administer the ligand or pharmaceutical composition of the invention, for example a ligand provided by e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The ligand or composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

The ligand or pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the ligand or pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule. A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a ligand or pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the ligand(s). Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

Exemplary TOIs

For example in any configuration, aspect, concept, example or configuration of the invention, the or each TOI is selected from the group consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AWl; AIGI; AKAP1; AKAP2; AIYIH; amyloid-beta; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; Axl; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAGI; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; C19orflO (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CB1; CCBP2 (D6/JAB61); CCL1 (I-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2 I eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3 (MIP-la); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8 (CMKBR8/TERI/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD7, CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CD96; CD207; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1/Cip1); CDKN1B (p27Kip1); CDKNIC; CDKN2A (p161NK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CELSR3; CERI; CHGA; CHGB; Chitinase; CHRNG; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CRLF2; CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYDi); CX3CR1 (V28); CXCR6; CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78 I LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR ISTRL33 I Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DAND5; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EphA4; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FCRL4; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEBI; GAGECI; Galectin-3; GALNAC4S-65T; GATA3; GDF5; GFI1; GGT1; GHR; GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GPR87; GPR137C; GRCC10 (C10); GRP; GSN (Gelsolin); GSTP1; HAVCR1; HAVCR2; HDAC4; EDAC5; HDAC7A; HDAC9; hepcidin; hemojuvelin; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOS; 1D2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; TFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL1IRA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; 1L13; IL13RA1; IL13RA2; 1L14; 1L15; IL15RA; IL16; 1L17; IL17B; IL17C; IL17R; 1L18; IL18BP; IL18R1; IL18RAP; 1L19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; 1L2; 1L20; IL2ORA; IL21R; 1L22; 1L22R; 1L22RA2; 1L23; 1L24; 1L25; 1L26; 1L27; 1L28A; 1L28B; 1L29; IL2RA; IL2RB; IL2RG; 1L3; 1L30; IL3RA; 1L4; IL4R; 1L5; IL5RA; 1L6; IL6 receptor; IL6ST (glycoprotein 130); 1L7; TL7R; 1L8; IL8RA; IL8RB; IL8RB; 1L9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; 1TGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAGI; JAKI; JAK3; JUN; K6HF; KAI1; KDR; MTLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAG3; LAMA5; LEP (leptin); LIGHT; Lingo-p75; Lingo-Troy; LPS; LRP5; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MK167 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-ifi); MTSS 1; MUC 1 (mucin); MYC; MYD88; NCK2; neurocan; Na_(v)1.7; Na_(v)1.8; NFKB 1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPG; OPRD1; OX40L; OX40; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PCSK9, PD-1, PD-L1; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; Placental Growth Factor (PIGF); PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNF11O (ZNF144); ROBO2; ROR1; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1; SERPINIA3; SERPINB5 (maspin); SERPINE1 (PAT-i); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Spri); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP1O; TDGF1; TEK; TGFA; TGFB1; TGFB1Il; TGFB2; TGFB3; TGFB1; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-i); TIM3; TMP3; tissue factor; TLR1O; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TMPRSS6; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSFlA; TNFRSFlB; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF1O (TRAIL); TNFSF1 1 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF1 5 (VEGI); TNFSF1 8; TNFSF4 (0X40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase lia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TRAIL; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGFA; VEGFB; VEGFC; versican; VHL C5; VLA-4; Wnt7A; XCL1 (lymphotactin); XCL2 (SCM-lb); XCR1 (GPR5/CCXCR1); YY1; and ZFPM2.

In an example, the TOI is a human TOI selected from Table 5.

In an example, the TOI is OX40 ligand.

In an example, the TOI is OX40.

In an example, the TOI is PCSK9.

In an example, the TOI is IL6 receptor (IL-6R).

In an example, the TOI is LIGHT.

In an example, the TOI is VEGF-A.

In an example, the TOI is TNF alpha.

In an example, the TOI is PIGF.

In an example, the TOI is IGF1R.

In an example, the TOI is OPG.

In an example, the TOI is ICOS

In an example, the TOI is NGF.

In an example, the TOI is BMP6.

In an example, the TOI is ferroportin.

In an example, the TOI is TMPRSS6.

In an example, the TOI is hemojuvelin.

In an example, the TOI is VEGF receptor.

In an example, the TOI is PDGF receptor.

In an example, the TOI is stem cell factor receptor.

In an example, the TOI is hepcidin.

In an example, the TOI is IL-4 receptor alpha.

In an example, the TOI is sclerostin.

In an example, the TOI is IL-13 receptor.

In an example, the TOI is CD7.

In an example, the TOI is delta-like ligand-4 (Dll4).

In an example, the TOI is HGF.

In an example, the TOI is angiopoietin-2 (Ang2).

In an example, the TOI is GDF8.

In an example, the TOI is ERBB3.

In an example, the TOI is IL-17 receptor.

In an example, the TOI is CD40.

In an example, the TOI is CD40 ligand.

In an example, the TOI is EGFR.

In an example, the TOI comprises a mutation as described herein for that TOI or is encoded by a nucleotide sequence comprising a SNP as described herein for that TOI. Optionally, additionally the human comprises said TOI comprising said mutation; or the human comprises a nucleotide sequence encoding said TOI comprising said mutation.

For example, the TOI is human PCSK9 and comprises a mutation I474, E670G, N425S or Q619P in SEQ ID NO: 1. Optionally, additionally the human comprises said PCSK9 comprising said mutation; or the human comprises a nucleotide sequence encoding said PCSK9 comprising said mutation. In an alternative, for example herein where a PCSK9 is stated to be of a specific “form”, eg, form f, c, r, p, e, h, aj or q, in the alternative the PCSK9 is a human PCSK9 comprising a mutation R46L, A53V, N425S, A443T, I474V, Q619P and E670G (eg, comprises a mutation I474, E670G, N425S or Q619P) in SEQ ID NO: 1; for example, the PCSK9 comprises I474V in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises E670G in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises Q619P in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises N425S in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises R46L in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg increasing cholesterol in the human); for example, the PCSK9 comprises A53V in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg increasing cholesterol in the human); for example, the PCSK9 comprises A443T in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg increasing cholesterol in the human).

For example, the TOI is human IL6R and comprises a mutation D358A or V3851 in SEQ ID NO: 78. Optionally, additionally the human comprises said IL6R comprising said mutation; or the human comprises a nucleotide sequence encoding said IL6R comprising said mutation.

For example, the TOI is human IL4Ra and comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Optionally, additionally the human comprises said IL4Ra comprising said mutation; or the human comprises a nucleotide sequence encoding said IL4Ra comprising said mutation.

For example, the TOI is huma Nav1.7 and comprises a mutation selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, eg, the mutation is 1161W. Optionally, additionally the human comprises said Nav1.7 comprising said mutation; or the human comprises a nucleotide sequence encoding said Nav1.7 comprising said mutation.

For example, the TOI is human VEGF-A and is encoded by a VEGF-A nucleotide sequence comprising a SNP selected from the group consisting of rs699947, rs833061, rs2010963, rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069, rs3025000 and rs1570360. Optionally, additionally the human comprises said VEGF-A comprising said mutation; or the human comprises a nucleotide sequence encoding said VEGF-A comprising said mutation.

For example, the TOI is human PDGF-B and is encoded by a PDGF-B nucleotide sequence comprising a SNP selected from the group consisting of rs142404523 (ie, a C corresponding to position −776) and a C at the position of rs1800818 (ie, a C corresponding to position −735). Optionally, additionally the human comprises said PDGF-B comprising said mutation; or the human comprises a nucleotide sequence encoding said PDGF-B comprising said mutation.

For example, the TOI is human PDGFR-B and is encoded by a PDGFR-B nucleotide sequence comprising a SNP selected from the group consisting of rs246395 (ie, a G corresponding to position 2601) and rs74943037 (ie, a T corresponding to position 1391). Optionally, additionally the human comprises said PDGFR-B comprising said mutation; or the human comprises a nucleotide sequence encoding said PDGFR-B comprising said mutation.

For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

The terms “decrease”, “reduced”, or “reduction” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” does not encompass a complete reduction as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder. However, for example, for the purposes of lowering or reducing cholesterol level, for example, a reduction by about 5-10 points can be considered a “decrease” or “reduction.”

In certain aspects of all embodiments of the invention, the term “inhibition” is used. Inhibition refers and refers to decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more including 100% inhibition as compared to a reference level. “Complete inhibition” refers to a 100% inhibition as compared to a reference level.

The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For the removal of doubt, “substantially” can refer to at least a 90% extent or degree of a characteristic or property of interest, e.g. at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or greater.

As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein. In some embodiments, the subject can be a non-human vertebrate, e.g. a primate, a rodent, a mouse, a rat, a pig, a sheep, a zebrafish, a frog, etc.

Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease or condition, e.g., a cardiovascular condition. A subject can be male or female.

A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

A “subject in need” or “human in need” of treatment for a particular condition can be a subject having that condition, such as increased cholesterol levels, diagnosed as having that condition, or at risk of developing that condition.

As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids with natural amino acids. When referring to “modified polypeptides” one refers to polypeptides that include modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins with the specified sequence. One can also use peptide homologs, peptide orthologs, peptide paralogs, peptide fragments and other equivalents, variants, fragments, and analogs of the peptides as these terms are understood by one of ordinary skill in the art.

As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA. In some aspects one can also use analogs of nucleic acids.

As used herein, the term “nucleic acid probe” refers to an isolated oligonucleotide molecule having a nucleic acid sequence which can hybridize to a target nucleic acid sequence, e.g. specifically hybridize to the target sequence. In some embodiments, a nucleic acid probe can further comprise a detectable label. In some embodiments, a nucleic acid probe can be attached to a solid surface. In some embodiments, a nucleic acid from is from about 5 nt to about 100 nt in length.

As used herein, the term “siRNA” refers to a nucleic acid that forms an RNA molecule comprising two individual strands of RNA which are substantially complementary to each other. Typically, the siRNA is at least about 15-40 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-40 nucleotides in length, and the double stranded siRNA is about 15-40 base pairs in length, preferably about 19-25 base nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In some embodiments, a siRNA can be blunt-ended. In some embodiments, a siRNA can comprise a 3′ and/or 5′ overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. The siRNA molecules can also comprise a 3′ hydroxyl group. In some embodiments, the siRNA can comprise a 5′ phosphate group. A siRNA has the ability to reduce or inhibit expression of a gene or target RNA when the siRNA is present or expressed in the same cell as the target gene, e.g. the target RNA. siRNA-dependent post-transcriptional silencing of gene expression involves cutting the target RNA molecule at a site guided by the siRNA.

As used herein, “PCSK9” or “proprotein convertase subtilisin/kexin type 9” refers to a serine protease involved in regulating the levels of the low density lipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah and Prat, 2007). PCSK9 has been shown to directly interact with the LDLR protein, be endocytosed along with the LDLR, and co-immunofluoresce with the LDLR throughout the endosomal pathway (Lagace et al., 2006). PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 2003). The sequence of PCSK9 for a variety of species is known, e.g., human PCSK9 (NCBI Gene ID No: 255738). Nucleotide and polypeptide sequences for a number of PCSK9 isoforms are provided herein, e.g., SEQ ID NOs: 1-37.

PCSK9 exists as both a pro-form and a mature form. Autocatalysis of the PCSK9 proform occurs between Gln152 and Ser153 (VFAQ|SIP (SEQ ID NO: 116)) (Naureckiene et al., 2003), and has been shown to be required for its secretion from cells (Seidah et al., 2003). The inactive form prior to this cleavage can be referred to herein as the “inactive”, “pro-form”, or “unprocessed” form of PCSK9. The C-terminal fragment generated by the autocatalysis event can be referred to herein as the “mature,” “cleaved”, “processed” or “active” PCSK9. Examples of pro-form and mature PCSK9 isoforms are provided herein, see, e.g. SEQ ID NOs: 1-27.

As used herein, the “catalytic domain” of PCSK9 refers to the portion of a PCSK9 polypeptide corresponding to positions 153 to 449 of PCSK9, e.g. of SEQ ID NO: 1. As used herein, the “C-terminal domain” of PCSK9 refers to the portion of a PCSK9 polypeptide corresponding to positions 450-692 of PCSK9, e.g., of SEQ ID NO: 1.

As used herein, a disease or condition “mediated by PCSK9” refers to a disease or condition which is caused by or characterized by a change in PCSK9, e.g. a change in expression level, a change in activity, and/or the presence of a variant or mutation of PCSK9. Non-limiting examples of such diseases or conditions can include, for example, a lipid disorder, hyperlipoproteinemia, hyperlipidemia; dyslipidemia; hypercholesterolemia, a heart attack, a stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, claudication (eg, claudication associated with elevated cholesterol), type II diabetes, high blood pressure, and a cardiovascular disease or condition. Another example is acute coronary syndrome. In an example, the disease or condition is an inflammatory or autoimmune disease or condition. Methods of identifying and/or diagnosing such diseases and conditions are well known to medical practitioners of ordinary skill.

A subject at risk of having or developing a disease or condition mediated by PCSK9 can be a subject exhibiting one or more signs or symptoms of such a disease or condition or having one or more risk factors for such a disease or condition, e.g. being overweight, having elevated cholesterol level, comprising one or more genetic polymorphisms known to predispose to the disease or condition, e.g., elevated cholesterol level, such as having a mutation in the LDLR (encoding low-density lipoprotein receptor) or APOB (encoding apolipoprotein B) or in the PCSK9 gene and/or having a family history of such a disease or condition.

As used herein, “ligand” refers to a molecule which can bind, e.g., specifically bind, to a second molecule or receptor. In some embodiments, a ligand can be, e.g., an antibody, antibody fragment, antibody portion, and/or affibody.

The term “variant” as used herein refers to a peptide or nucleic acid that differs from the polypeptide or nucleic acid (eg, the most common one in humans, eg, most frequent in a database as disclosed herein, such as the 1000 Genomes Project database) by one or more amino acid or nucleic acid deletions, additions, yet retains one or more specific functions or biological activities of the naturally occurring molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring amino acid residue. Such substitutions may be classified as “conservative”, in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention may also be “non-conservative”, in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid. In some embodiments amino acid substitutions are conservative. Also encompassed within the term variant when used with reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide).

Variants of PCSK9 are provided elsewhere herein. Variants of PCSK9 can include the forms described herein as a, f, c, r, p, m, e h, aj, and q. Sequences of these variants are provided herein, see, e.g, SEQ ID NOs:1-27 and in Table 1, 2 or 6.

In some aspects, one can use “synthetic variants”, “recombinant variants”, or “chemically modified” polynucleotide variants or polypeptide variants isolated or generated using methods well known in the art. “Modified variants” can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Some aspects use include insertion variants, deletion variants or substituted variants with substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins. The term “conservative substitution,” when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity. For example, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties. Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

“Conservative amino acid substitutions” result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Thus, a “conservative substitution” of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not reduce the activity of the peptide, (i.e. the ability of the peptide to penetrate the blood brain barrier (BBB)). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered “conservative substitutions” if the change does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents.

In alternative embodiments, one can select the amino acid which will substitute an existing amino acid based on the location of the existing amino acid, i.e. its exposure to solvents (i.e. if the amino acid is exposed to solvents or is present on the outer surface of the peptide or polypeptide as compared to internally localized amino acids not exposed to solvents). Selection of such conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. MoI Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol. 119(1986); 205-218 and S. French and B. Robson, J. MoI. Evol. 19(1983)171. Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent), for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acid substitutions encompassed suitable for amino acids on the interior of a protein or peptide, for example one can use suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent), for example but not limited to, one can use the following conservative substitutions: where Y is substituted with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term of variants.

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria. Antibodies can be humanized using routine technology.

As described herein, an “antigen” is a molecule that is bound by a binding site on an antibody agent. Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. The term “antigenic determinant” refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody fragment” refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody fragment can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody fragment can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody fragment” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.

As used herein, “antibody variable domain” refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2, and CDR3), and Framework Regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. According to the methods used in this invention, the amino acid positions assigned to CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or according to IMGT nomenclature.

D domain or region refers to the diversity domain or region of an antibody chain. J domain or region refers to the joining domain or region of an antibody chain.

An antibody “gene segment”, e.g. a VH gene segment, D gene segment, or JH gene segment refers to oligonucleotide having a nucleic acid sequence that encodes that portion of an antibody, e.g. a VH gene segment is an oligonucleotide comprising a nucleic acid sequence that encodes a polypeptide VH domain.

The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FR”). The extent of the framework region and CDRs has been precisely defined (see, IMGT or Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, which are used interchangeably herein are used to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546; which is incorporated by reference herein in its entirety), which consists of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality.

As used herein, the term “antibody binding site” refers to a polypeptide or domain that comprises one or more CDRs of an antibody and is capable of binding an antigen. For example, the polypeptide comprises a CDR3 (eg, HCDR3). For example the polypeptide comprises CDRs 1 and 2 (eg, HCDR1 and 2) or CDRs 1-3 of a variable domain of an antibody (eg, HCDRs1-3). In an example, the antibody binding site is provided by a single variable domain (eg, a VH or VL domain). In another example, the binding site comprises a VH/VL pair or two or more of such pairs.

As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. For example, in an diagnostic test the specific binding of a ligand can distinguish between two variant PCSK9 proteins as described herein. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. In the context of oligonucleotide strands which interact via hybridization, specific binding can be “specific hybridization.”

Additionally, and as described herein, a recombinant human(ized) antibody can be further optimized to decrease potential immunogenicity, while maintaining functional activity, for therapy in humans. In this regard, functional activity means a polypeptide capable of displaying one or more known functional activities associated with a recombinant antibody or antibody reagent thereof as described herein. Such functional activities include, e.g. the ability to bind to a target molecule.

The term “immunizing” refers to the step or steps of administering one or more antigens to an animal so that antibodies can be raised in the animal Generally, immunizing comprises injecting the antigen or antigens into the animal. Immunization can involve one or more administrations of the antigen or antigens. Suitable methods are prime-boost and RIMMS procedures as known to the skilled person in the art.

As used herein, an “affibody” refers to a relatively small synthetic protein molecule that has high binding affinity for a target protein (e.g. for PCSK9 or a variant thereof). Affibodies are composed of a three-helix bundle domain derived from the IgG-binding domain of staphylococcal protein A. The protein domain consists of a 58 amino acid sequence, with 13 randomized amino acids affording a range of affibody variants. Despite being significantly smaller than an antibody (an affibody weighs about 6 kDa while an antibody commonly weighs about 150 kDa), an affibody molecule works like an antibody since its binding site is approximately equivalent in surface area to the binding site of an antibody.

As used herein, “VH3-23*04” refers to a human VH domain variant comprising the polypeptide sequence of SEQ ID NO: 38. As opposed to the reference sequence, VH3-23*04 has a valine residue instead of a leucine residue (see FIGS. 3 and 4 ; L24V, numbering including signal sequence; valine at position 5 shown in FIG. 4 ) as a result of the presence of the rs56069819 SNP in the nucleic acid sequence encoding the VH domain. As used herein, “rs56069819” refers to a mutation or variant in a VH gene segment from adenosine to cytosine (or thymine to guanine, depending upon the strand of DNA which is being read), resulting in the VH domain encoding VH3-23*04. Rs56069819 is depicted in FIG. 4 and SEQ ID NO: 39, which demonstrate the T->G mutation (it is noted that the dbSNP entry for RS5606819 depicts the other strand, which comprises the A->C mutation). Further description of VH3-23*04 can be found, e.g., in US Patent Publication 2013/0071405; which is incorporated by reference herein in its entirety.

As used herein, “determine” or “determining” refers to ascertaining, e.g., by a quantitative or qualitative analysis. As used herein, “has been determined” can refer to ascertaining on the basis of previously obtained information or simultaneously obtained information.

In some aspects of all embodiments of the invention selecting can include automation such as a computer implemented software program that upon input of the relevant data such as ethnicity or a panel of SNP data can make the determination based on the instructions set forth herein.

As used herein, “assaying” refers to assessing, evaluating, quantifying, measuring, or characterizing an analyte, e.g., measuring the level of an analyte in a sample, identifying an analyte, or detecting the presence or absence of an analyte in a sample. In some embodiments, assaying refers to detecting a presence or absence of the analyte of interest. In some embodiments, assaying refers to quantifying an amount of an analyte, e.g., providing a measure of concentration or degree of analyte abundance. In some embodiments, assaying refers to enumerating the number of molecules of analyte present in a sample and/or specimen, e.g., to determine an analyte copy number.

As used herein “multiplex” refers to the carrying out of a method or process simultaneously and in the same reaction vessel on two or more, typically three or more, different target sequences, e.g. on two or more isoforms of PCSK9, or PCSK9 and an additional target. A multiplex analysis typically includes analysis of 10-50; 10-100; 10-1000, 10-5000, 10-10000 reactions in a multiplex format, such as a multiwall, an array, or a multichannel reaction.

Often the analysis or multiplex analysis is also automated using robotics and typically software executed by a computer and may include a robotic handling of samples. automatic or robotic selection of positive or negative results, assaying for presence of absence of a target, such as a nucleic acid polymorphism or a protein variant.

The term “biological sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a sample from a subject. Exemplary biological samples include, but are not limited to, a biofluid sample; serum; plasma; urine; saliva; hair, epithelial cells, skin, a tumor biopsy and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” or “biological sample” also includes untreated or pretreated (or pre-processed) biological samples. For the analysis of nucleic acids, the biological sample should typically comprise at least one cell comprising nucleic acids.

The test sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells (e.g. isolated at a prior time point and isolated by the same or another person). In addition, the test sample can be freshly collected or a previously collected, refrigerated, frozen or otherwise preserved sample.

In some embodiments, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.

As used herein, “genotyping” refers to a process of determining the specific allelic composition of a cell and/or subject at one or more position within the genome, e.g. by determining the nucleic acid sequence at that position. Genotyping refers to a nucleic acid analysis and/or analysis at the nucleic acid level. As used herein, “phenotyping” refers a process of determining the identity and/or composition of an expression product of a cell and/or subject, e.g. by determining the polypeptide sequence of an expression product. Phenotyping refers to a protein analysis and/or analysis at the protein level.

As used herein, the term “nucleic acid amplification” refers to the production of additional copies of a nucleic acid sequence and is typically carried out using polymerase chain reaction (PCR) or ligase chain reaction (LCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N. Y.). Other methods for amplification are also contemplated in aspects of the invention.

The term “allele-specific amplification” refers to a reaction (e.g., PCR reaction) in which at least one of the primers (e.g., allele-specific primer) is chosen from a polymorphic area of gene (e.g., single nucleotide polymorphism), with the polymorphism located at or near the primer's 3′-end. A mismatched primer will not initiate amplification, whereas a matched primer will initiate amplification. The appearance of an amplification product is indicative of the presence of the polymorphism.

As used herein, “sequencing” refers to the determination of the exact order of nucleotide bases in a strand of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) or the exact order of amino acids residues or peptides in a protein. Nucleic acid sequencing can be done using Sanger sequencing or next-generation high-throughput sequencing.

As used herein “next-generation sequencing” refers to oligonucleotide sequencing technologies that have the capacity to sequence oligonucleotides at speeds above those possible with conventional sequencing methods (e.g. Sanger sequencing), due to performing and reading out thousands to millions of sequencing reactions in parallel. Non-limiting examples of next-generation sequencing methods/platforms include Massively Parallel Signature Sequencing (Lynx Therapeutics); 454 pyro-sequencing (454 Life Sciences/Roche Diagnostics); solid-phase, reversible dye-terminator sequencing (Solexa/Illumina): SOLiD technology (Applied Biosystems); Ion semiconductor sequencing (ION Torrent); DNA nanoball sequencing (Complete Genomics); and technologies available from Pacific Biosciences, Intelligen Bio-systems, Oxford Nanopore Technologies, and Helicos Biosciences. Next-generation sequencing technologies and the constraints and design parameters of associated sequencing primers are well known in the art (see, e.g. Shendure, et al., “Next-generation DNA sequencing,” Nature, 2008, vol. 26, No. 10, 1135-1145; Mardis, “The impact of next-generation sequencing technology on genetics,” Trends in Genetics, 2007, vol. 24, No. 3, pp. 133-141; Su, et al., “Next-generation sequencing and its applications in molecular diagnostics” Expert Rev Mol Diagn, 2011, 11(3):333-43; Zhang et al., “The impact of next-generation sequencing on genomics”, J Genet Genomics, 2011, 38(3):95-109; (Nyren, P. et al. Anal Biochem 208: 17175 (1993); Bentley, D. R. Curr Opin Genet Dev 16:545-52 (2006); Strausberg, R. L., et al. Drug Disc Today 13:569-77 (2008); U.S. Pat. Nos. 7,282,337; 7,279,563; 7,226,720; 7,220,549; 7,169,560; 6,818,395; 6,911,345; US Pub. Nos. 2006/0252077; 2007/0070349; and 20070070349; which are incorporated by reference herein in their entireties).

As used herein, “nucleic acid hybridization” refers to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands. In some embodiments, nucleic acid hybridization can refer to a method of determining a nucleic acid sequence and/or identity by hybridizing a nucleic acid sample with a probe, e.g. Northern or Southern blot analysis or microarray analysis.

As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). For treatment to be effective a complete cure is not contemplated. The method can in certain aspects include cure as well.

As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.

Multiple compositions can be administered separately or simultaneously. Separate administration refers to the two compositions being administered at different times, e.g. at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One can also administer compositions at 24 hours apart, or even longer apart. Alternatively, two or more compositions can be administered simultaneously, e.g. less than 10 or less than 5 minutes apart. Compositions administered simultaneously can, in some aspects, be administered as a mixture, with or without similar or different time release mechanism for each of the components.

As used herein, “contacting” refers to any suitable means for delivering, or exposing, an agent to at least one complex, enzyme, or cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.

As used herein, “obtain” refers to any method of acquiring, securing, procuring, or coming into the possession of, e.g. a sample. Obtaining a biological sample from a subject can comprise physical removing a sample from a subject (e.g. drawing blood or taking a hair or saliva sample) without or without active participation from the subject; receiving a sample from a subject (e.g. the subject collects a saliva or hair sample themselves and provides it, e.g. in a container provided for the purpose); or procuring a sample from a storage facility, medical facility, or medical provider. Obtain from the human or subject, refers to an active step of, e.g., drawing blood or taking a tissue or cell sample.

As used herein, “cholesterol level” refers to a level of one or more of total cholesterol, LDL cholesterol, HDL cholesterol, and/or triglycerides. Cholesterol levels can be the level of cholesterol in the blood of a subject.

As used herein in reference to cholesterol levels, “maintain” refers to preventing the level from worsening (e.g. increasing). In some embodiments, maintaining a particular level refers to a process that results in the cholesterol level not increasing by more than 10% over time. Maintaining may also refer to maintaining a previously achieved level. For example, if a human has received statin treatment, one can maintain the cholesterol level achieved using the statin treatment.

In some embodiments, the subject treated according to the methods described herein has previously had their cholesterol level reduced. As used herein, “previously reduced” indicates that at a prior point in time, the subject experienced a decrease in cholesterol levels. The decrease can be due to administration of a pharmaceutical composition (e.g. administration of a composition as described herein or another composition, e.g. a statin) or due to another cause, e.g. a change in diet and/or exercise.

An existing treatment for high cholesterol levels is the administration of a statin. As referred to herein, a “statin” (also known as HMG-CoA reductase inhibitors) are inhibitors of the enzyme HMG-coA reductase, which mediates cholesterol production in the liver. Statins, by competitively binding HMG-CoA reductase, prevent the binding of HMG-CoA to the enzyme and thereby inhibit the activity of the reductase (e.g. the production of mevalonate). Non-limiting examples of statins can include atorvastatin (LIPITOR™), fluvastatin (LESCOLI™), lovastatin (MEVACOR™, ALTOCOR™), pitavastatin (LIVALO™), pravastatin (PRAVACHOL™), rosuvastatin (CRESTOR™), and simvastatin (ZOCOR™). Statins can be administered in combination with other agents, e.g. the combination of ezetimibe and simvastatin.

Some subjects are, or become, resistant to statin treatment. As used herein, “resistant to statin treatment” or “reduced responsiveness to statin treatment” refers to a subject exhibiting a statistically significantly lower response to the administration of a statin as compared to a reference level. The reference level can be, e.g., the average response for a population of subjects or the level of the individual subject at an earlier date. A response to statin treatment is readily measured by one of skill in the art, e.g., measurement of cholesterol levels, changes in cholesterol levels, and/or HMG-CoA reductase activity.

As used herein, the term “detectable label” refers to a molecule or moiety that can be detected, e.g. measured and/or determined to be present or absent. Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.

In some embodiments, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

In other embodiments, the detectable label can be a fluorescent compound. When the fluorescently label is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6-carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J), N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments, a detectable label can be a radiolabel including, but not limited to ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and ³³P. In some embodiments, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use as a detectable label can include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

In some embodiments, reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, Calif. A reagent can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

As used herein, “authorization number” or “marketing authorization number” refers to a number issued by a regulatory agency upon that agency determining that a particular medical product and/or composition may be marketed and/or offered for sale in the area under the agency's jurisdiction. As used herein “regulatory agency” refers to one of the agencies responsible for evaluating, e.g, the safety and efficacy of a medical product and/or composition and controlling the sales/marketing of such products and/or compositions in a given area. The Food and Drug Administration (FDA) in the US and the European Medicines Agency (EPA) in Europe are but two examples of such regulatory agencies. Other non-limiting examples can include SDA, MPA, MHPRA, IMA, ANMAT, Hong Kong Department of Health-Drug Office, CDSCO, Medsafe, and KFDA.

As used herein, “injection device” refers to a device that is designed for carrying out injections, an injection including the steps of temporarily fluidically coupling the injection device to a person's tissue, typically the subcutaneous tissue. An injection further includes administering an amount of liquid drug into the tissue and decoupling or removing the injection device from the tissue. In some embodiments, an injection device can be an intravenous device or IV device, which is a type of injection device used when the target tissue is the blood within the circulatory system, e.g., the blood in a vein. A common, but non-limiting example injection device is a needle and syringe.

As used herein, a “buffer” refers to a chemical agent that is able to absorb a certain quantity of acid or base without undergoing a strong variation in pH.

As used herein, “packaging” refers to how the components are organized and/or restrained into a unit fit for distribution and/or use. Packaging can include, e.g., boxes, bags, syringes, ampoules, vials, tubes, clamshell packaging, barriers and/or containers to maintain sterility, labeling, etc.

As used herein, “instructions” refers to a display of written, printed or graphic matter on the immediate container of an article, for example the written material displayed on a vial containing a pharmaceutically active agent, or details on the composition and use of a product of interest included in a kit containing a composition of interest. Instructions set forth the method of the treatment as contemplated to be administered or performed.

As used herein, a “solid surface” refers to an object suitable for the attachment of biomolecules. Non-limiting examples of a solid surface can include a particle (including, but not limited to an agarose or latex bead or particle or a magnetic particle), a bead, a nanoparticle, a polymer, a substrate, a slide, a coverslip, a plate, a dish, a well, a membrane, and/or a grating. The solid surface can include many different materials including, but not limited to, polymers, plastics, resins, polysaccharides, silicon or silica based materials, carbon, metals, inorganic glasses, and membranes.

As used herein, “classification” of a subject, e.g., classification of the subject's ancestry refers to determining if the subject has biological ancestors who originated in a particular geographical area, and are therefore likely to have particular genetic variants found in the populations which have historically occupied that area. Classification can comprise, e.g. obtaining information on the subject's family, interviewing the subject or a family member regarding their biological family's ancestry, and/or genetic testing. Classification can be on the basis used for the 1000 Genomes Project, as will be familiar to the skilled person in the art. In some embodiments, the subject can be classified as being of a particular ancestry if at least the subject's genome comprises a substantial number of different alleles in common with other humans of that ancestry (eg, determined by reference to the 1000 Genomes Project database), for example, at least 10, 20, 30, 40, 50 or 100 or more alleles in common. Abbreviations for particular ancestral groups are provided in Table 4.

The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.

Other terms are defined herein within the description of the various aspects of the invention.

All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

It will be understood that particular configurations, aspects, examples, clauses and embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps

Any part of this disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.

References to sequences by “SEQ ID NO:” herein (eg, in the claims) is by reference to the sequences appearing in the Tables; similarly reference to position numbers “in SEQ ID NO:” (eg, in the claims) is by reference to the sequences appearing in the Tables herein (eg, as per Tables 6, 10 and 17). Thus, for example, “an Asp corresponding to position 204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42” refers to the Asp and Leu labelled as being at positions 204 and 206 respectively of SEQ ID NO: 42 shown in Table 10 herein.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

The present invention is described in more detail in the following non limiting Examples.

Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

1. A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising

-   -   a. selecting a human that is positive for the TOI polymorphic         variant, wherein the TOI in said human is encoded by a         nucleotide sequence comprising an allele having a cumulative         human allele frequency of less than 50% and/or wherein the TOI         in said human is encoded by a nucleotide sequence comprising an         allele having total human genotype frequency of less than 50%;         and     -   b. administering to the human an anti-TOI ligand to target the         TOI in the human to treat or prevent said disease or condition.

In a first alternative, paragraph 1 provides:—

A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising comprising administering to said human an anti-TOI ligand (eg, an antibody or antibody fragment) that specifically binds a TOI variant that is encoded by a nucleotide sequence comprising a SNP having a cumulative human allele frequency of less than 50% (eg, less than 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2, or 1%) and/or wherein the TOI variant is encoded by a nucleotide sequence comprising a SNP having total human genotype frequency of less than 50% (eg, less than 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2, or 1%); wherein the human comprises a (eg, said) TOI-encoding nucleotide sequence comprising said SNP; wherein

(i) the ligand (eg, antibody or fragment) comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment comprising a SNP, and wherein the human comprises a (eg, said) VH segment comprising said SNP; or (ii) the ligand (eg, antibody or fragment) comprises a Vλ domain derived from the recombination of a human Vλ segment and a human Jλ segment, the human Vλ segment comprising a SNP, and wherein the human comprises a (eg, said) VW segment comprising said SNP; or (iii) the ligand (eg, antibody or fragment) comprises a Vκ domain derived from the recombination of a human Vκ segment and a human Jκ segment, the human Vκ segment comprising a SNP, and wherein the human comprises a (eg, said) Vκ segment comprising said SNP; or (iv) the ligand (eg, antibody or fragment) comprises a heavy chain constant region (eg, a gamma-1, 2, 3 o 4 constant region) encoded by a nucleotide sequence comprising a SNP, and wherein the human comprises a (eg, said) heavy chain constant region (eg, a gamma-1, 2, 3 o 4 constant region respectively) comprising said SNP; or (v) the ligand (eg, antibody or fragment) comprises a lambda chain constant encoded by a nucleotide sequence comprising a SNP, and wherein the human comprises a (eg, said) lambda chain constant region comprising said SNP; or (vi) the ligand (eg, antibody or fragment) comprises a kappa chain constant encoded by a nucleotide sequence comprising a SNP, and wherein the human comprises a (eg, said) kappa chain constant region comprising said SNP.

In a second alternative, paragraph 1 provides:—

A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising comprising administering to said human an anti-TOI ligand (eg, an antibody or antibody fragment) that specifically binds a TOI variant that comprises a single amino acid variation (ie, a single amino acid at a position where there is difference between TOI variants in humans) having a cumulative human allele frequency of less than 50% (eg, less than 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2, or 1%) and/or wherein the amino acid variant has a total human genotype frequency of less than 50% (eg, less than 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2, or 1%); wherein the human expresses TOI proteins comprising said single amino acid variation; wherein

(i) the ligand (eg, antibody or fragment) comprises a VH domain comprising the same single amino acid variation, and wherein the human expresses VH domains comprising said single amino acid variation; or (ii) the ligand (eg, antibody or fragment) comprises a Vλ domain comprising a single amino acid variation, and wherein the human expresses Vλ domains comprising the same single amino acid variation; or (iii) the ligand (eg, antibody or fragment) comprises a Vκ domain comprising a single amino acid variation, and wherein the human expresses Vκ domains comprising the same single amino acid variation; or (iv) the ligand (eg, antibody or fragment) comprises a heavy chain constant region (eg, a gamma-1, 2, 3 o 4 constant region, eg, wherein the constant region is an Fc, CH1, CH2 or CH3) comprising a single amino acid variation, and wherein the human expresses a heavy chain constant region (eg, a gamma-1, 2, 3 o 4 constant region, eg, wherein the constant region is an Fc, CH1, CH2 or CH3 respectively) comprising the same single amino acid variation; or (v) the ligand (eg, antibody or fragment) comprises a lambda chain constant region comprising a single amino acid variation, and wherein the human expresses a lambda chain constant region comprising the same single amino acid variation; or (vi) the ligand (eg, antibody or fragment) comprises a kappa chain constant region comprising a single amino acid variation, and wherein the human expresses a kappa chain constant region comprising the same single amino acid variation.

The term “comprises a single amino acid variation” includes the possibility of there being further amino acid variations in the TOI sequence.

In an example, frequencies herein are according to the 1000 Genomes database, eg, the Phase I database or as shown in Ensembl (available on the world wide web at ensembl.org), eg, at 1 Oct. 2013 or 1 Oct. 2014.

In an example, each SNP encodes a non-synonymous amino acid variation, ie, the SNP is not a silent mutation.

Optionally, the method comprises selecting said human comprising the nucleotide sequence prior to administration.

In a third alternative where the TOI is human PCSK9, paragraph 1 provides:—

A method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation as defined herein (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding a valine at the amino acid corresponding to position 5 of SEQ ID NO: 40 and wherein said human comprises (i) a VH gene segment encoding the framework 1 of SEQ ID NO: 40 and (ii) a nucleotide sequence encoding a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation in SEQ ID NO: 1.

In a fourth alternative where the TOI is human PCSK9, paragraph 1 provides:—

A method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation as defined herein (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody comprises a VL domain derived from the recombination of a human VL segment and a human JL segment, the human VL segment is a Vλ or Vκ disclosed herein and wherein said human comprises (i) said VL gene segment and (ii) a nucleotide sequence encoding a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation in SEQ ID NO: 1.

In a fifth alternative where the TOI is human PCSK9, paragraph 1 provides:—

A method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation as defined herein (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody comprises a C domain encoded by a human CH, Cλ or Cκ gene segment disclosed herein and wherein said human comprises (i) said C gene segment and (ii) a nucleotide sequence encoding a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation in SEQ ID NO: 1.

The following paragraphs 2-8, 19 and 49 onwards relate to any of the alternatives of paragraph 1.

-   2. The method of paragraph 1, wherein before step (a) the ligand has     been or is determined as being capable of specifically binding to     said TOI variant. -   3. The method of paragraph 1 or 2, comprising determining that the     human is positive for the TOI polymorphic variant, optionally     wherein the step of determining comprises determining that the human     is positive for a nucleotide variant encoding said TOI variant. -   4. The method of paragraph 3, wherein the step of determining     comprises assaying a biological sample obtained from said human for     a nucleotide polymorphism encoding said TOI polymorphic variant. -   5. The method of paragraph 3 or 4, wherein the step of determining     comprises assaying a biological sample obtained from said human for     a protein corresponding to the TOI polymorphic variant. -   6. The method of any preceding paragraph, wherein said frequency is     less than 15%. -   7. The method of any preceding paragraph, wherein said frequency is     less than 10%. -   8. The method of any preceding paragraph, wherein the ligand is     capable of specifically binding to two or more different TOI     variants, each being encoded by a nucleotide sequence comprising an     allele having a cumulative human allele frequency of less than 50%     and/or having a total human genotype frequency of less than 50%. -   9. A method of treating or preventing a disease or condition in a     human, wherein the disease or condition is mediated by a Target of     Interest (TOI), wherein the TOI is present in humans as different     polymorphic variants, the method comprising     -   a. selecting a human that is negative for a variant nucleotide         sequence comprising an allele having a cumulative human allele         frequency of less than 50% and/or having a total human genotype         frequency of less than 50%; or that the human is negative for a         TOI variant encoded by a nucleotide sequence comprising the         allele having a cumulative human allele frequency of less than         50% and/or having a total human genotype frequency of less than         50%; and     -   b. administering to the human an anti-TOI ligand to target the         TOI variant in the human and treat or prevent said disease or         condition, wherein the TOI in said human is a variant encoded by         a nucleotide sequence comprising an allele having a cumulative         human allele frequency of more than 50% and/or having a total         human genotype frequency of more than 50%. -   10. The method of paragraph 9, comprising determining that the human     is positive for the TOI polymorphic variant, optionally wherein the     determining comprises that the human has been or is phenotyped as     positive for the most frequent TOI variant or genotyped for the     nucleotide sequence thereof. -   11. The method of paragraph 10, wherein determining comprises     assaying for the nucleotide sequence to determine the presence of     said allele. -   12. The method of paragraph 11, wherein the assaying comprises     nucleic acid amplification. -   13. The method of paragraph 11 or 12, wherein the assaying comprises     hybridization, sequencing, or next generation sequencing. -   14. The method of any of paragraphs 11-13, further comprising the     step of obtaining a biological sample from the human. -   15. The method of any one of paragraphs 9-14, wherein the ligand has     been or is determined as being capable of specifically binding to     the most frequent TOI variant. -   16. The method of any one of paragraphs 9-15, wherein the ligand has     been or is determined as being substantially incapable of     neutralising or inhibiting said TOI variant. -   17. The method of any one of paragraphs 9-16, wherein the ligand is     capable of specifically binding to the most frequent TOI variant. -   18. The method of any one of paragraphs 9-17, wherein the ligand is     capable of specifically binding to two or more different TOI     variants, each being encoded by a nucleotide sequence comprising an     allele having a cumulative human allele frequency of more than 50%. -   19. The method of any preceding paragraph, wherein said TOI     polymorphic variant has been or is determined as being present in at     least two different human ethnic populations. -   20. The method of any preceding paragraph, wherein said cumulative     human allele frequency is the frequency in a database of     naturally-occurring sequences sampled from at least 15 different     human ethnic populations and comprising at least 1000 sequences. -   21. The method of any of the preceding paragraphs, wherein the     ligand is an antibody, antibody fragment or an affibody. -   22. The method of any of the preceding paragraphs, wherein the     ligand comprises a nucleotide sequence that specifically hybridises     to a TOI nucleotide sequence comprising an allele having a     cumulative human allele frequency of less than 50% and/or having a     total human genotype frequency of less than 50% or an RNA transcript     thereof; and/or the ligand comprises a nucleotide sequence that     comprises at least 10 contiguous nucleotides of a nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or having a total human genotype frequency of less than 50% or     is an antisense sequence thereof. -   23. The method of any of the preceding paragraphs, wherein the     genome of said human comprises an allele having a cumulative human     allele frequency of less than 50% and the allele is found in at     least 2 different ethnic populations. -   24. A composition comprising a ligand capable of binding a target of     interest encoded by a nucleotide sequence comprising an allele     having a cumulative human allele frequency of less than 50% and the     allele is found in at least 2 different ethnic populations and     optionally a pharmaceutically acceptable carrier and optionally a     label or instructions for use to treat and/or prevent said disease     or condition in a human; optionally wherein the label or     instructions comprise a marketing authorisation number issued by a     regulatory authority. -   25. A kit for treating or preventing a condition or disease mediated     by a target of interest as recited in any preceding paragraph, the     kit comprising a ligand capable of specifically binding a target of     interest encoded by a nucleotide sequence comprising an allele     having a cumulative human allele frequency of less than 50% and the     allele is found in at least 2 different ethnic populations; and     optionally in combination with a label or instructions for use to     treat and/or prevent said disease or condition in a human;     optionally wherein the label or instructions comprise a marketing     authorisation number issued by a regulatory agency; optionally     wherein the kit comprises an injection pen or IV container that     comprises the ligand. -   26. The composition of paragraph 24 or the kit of paragraph 25,     wherein the regulatory agency is FDA or EMA. -   27. A method of producing an anti-human TOI antibody binding site,     the method comprising obtaining a plurality of anti-TOI antibody     binding sites, screening the antibody binding sites for binding to a     TOI comprising an amino acid sequence encoded by a nucleotide     sequence comprising an allele having a cumulative human allele     frequency of less than 50% and/or a total human genotype frequency     of less than 50%, or to a peptide thereof that comprises an amino     acid variation from the corresponding sequence encoded by the     TOI-encoding nucleotide sequence comprising an allele having the     highest cumulative human allele frequency and/or the highest total     human genotype frequency, and isolating an antibody binding site     that binds in the screening step. -   28. A method of producing an anti-human TOI antibody, the method     comprising immunising a non-human vertebrate with a TOI comprising     an amino acid sequence encoded by a nucleotide sequence comprising     an allele having a cumulative human allele frequency of less than     50% and/or a total human genotype frequency of less than 50%, or to     a peptide thereof that comprises an amino acid variation from the     corresponding sequence encoded by the TOI-encoding nucleotide     sequence comprising an allele having the highest cumulative human     allele frequency and/or the highest total human genotype frequency,     and isolating an antibody that binds a TOI comprising an amino acid     sequence encoded by a TOI nucleotide sequence comprising an allele     having a cumulative human allele frequency of less than 50% and/or a     total human genotype frequency of less than 50%, and optionally     producing a TOI-binding fragment or derivative of the isolated     antibody. -   29. The method of paragraph 28, wherein the non-human vertebrate is     a mouse or a rat. -   30. The method of paragraph 29 or 30, comprising the step of     obtaining a nucleic acid encoding the antibody, fragment, derivative     or binding site and optionally inserting the nucleic acid in an     expression vector. -   31. A kit for TOI genotyping a human, wherein the kit comprises a     nucleic acid comprising a nucleotide sequence that specifically     hybridises to a TOI nucleotide sequence selected having a cumulative     human allele frequency of less than 50% and/or a total human     genotype frequency of less than 50% or an RNA transcript thereof;     and/or the nucleic acid comprises a nucleotide sequence that     comprises at least 10 contiguous nucleotides of a TOI nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or a total human genotype frequency of less than 50% or is an     antisense sequence thereof. -   32. A kit for TOI genotyping or phenotyping a human, wherein the kit     comprises a ligand capable of binding a target of interest encoded     by a nucleotide sequence comprising an allele having a cumulative     human allele frequency of less than 50% or an antibody, fragment or     derivative produced by the method of any one of paragraphs 28 to 30. -   33. The kit of paragraph 32, wherein the allele is found in at least     2 different ethnic populations. -   34. Use of an anti-TOI ligand that specifically binds a human TOI     comprising an amino acid sequence encoded by a TOI nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or a total human genotype frequency of less than 50%, in the     manufacture of a medicament for treating and/or preventing a     TOI-mediated disease or condition in a human whose genome comprises     a TOI nucleotide sequence having a cumulative human allele frequency     of less than 50% and/or having a total human genotype frequency of     less than 50%. -   35. Use of an anti-TOI ligand that specifically binds a human TOI     comprising an amino acid sequence encoded by a TOI nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or a total human genotype frequency of less than 50%, in the     manufacture of a medicament for targeting said TOI in a human to     treat and/or prevent a disease or condition mediated by TOI. -   36. A method of targeting a Target of Interest (TOI) for treating     and/or preventing a TOI-mediated disease or condition in a human,     the method comprising administering an anti-TOI ligand to a human     comprising a TOI nucleotide sequence comprising an allele selected     as having a cumulative human allele frequency of less than 50%     and/or a total human genotype frequency of less than 50%, whereby a     TOI encoded by said nucleotide sequence is targeted. -   37. The method of paragraph 36, wherein the method comprises     targeting a human TOI comprising an amino acid sequence with said     ligand to treat and/or prevent said disease or condition in said     human, wherein said amino acid sequence is encoded by a nucleotide     sequence comprising an allele having a cumulative human allele     frequency of less than 50% and/or a total human genotype frequency     of less than 50%. -   38. A method of Target of Interest (TOI) genotyping a nucleic acid     sample of a human, the method comprising assaying in the sample the     presence of a TOI nucleotide sequence comprising an allele having a     cumulative human allele frequency of less than 50% and/or having a     total human genotype frequency of less than 50%. -   39. A method of Target of Interest (TOI) typing a protein sample of     a human, the method comprising assaying the sample the presence of a     TOI amino acid sequence encoded by a TOI nucleotide sequence     comprising an allele having a cumulative human allele frequency of     less than 50% and/or having a total human genotype frequency of less     than 50%. -   40. The method of paragraph 38 or 39, comprising obtaining a sample     of serum, blood, faeces, hair, urine or saliva from a human, whereby     the nucleic acid or protein sample is obtained for use in the step     of assaying said sequence. -   41. The method of any one of paragraphs 38-40, comprising using a     ligand capable of targeting a nucleic acid sequence comprising an     allele having a cumulative human allele frequency of less than 50%     and/or having a total human genotype frequency of less than 50% or a     ligand capable of specifically binding the TOI encoded by said     nucleic acid sequence to carry out said identifying step. -   42. A diagnostic kit comprising a ligand that is capable of binding     a human Target of Interest (TOI) comprising an amino acid sequence     encoded by a TOI nucleotide sequence comprising an allele having a     cumulative human allele frequency of less than 50% and/or a total     human genotype frequency of less than 50% and instructions for     carrying out the method of any one of paragraphs 38-41. -   43. The diagnostic kit wherein the ligand is selected from an     antibody, antibody fragment, antibody portion, affybody,     oligonucleotide, modified oligonucleotide, antisense     oligonucleotide, siRNA, and microRNA. -   44. A diagnostic kit comprising a nucleic acid probe comprising a     nucleotide sequence that specifically hybridises a Target of     Interest (TOI) nucleotide sequence comprising an allele having a     cumulative human allele frequency of less than 50% and/or a total     human genotype frequency of less than 50% or an RNA transcript     thereof and instructions for carrying out the method of paragraph 38     or 39. -   45. The method, ligand, composition, kit or use of any preceding     paragraph, wherein the TOI is encoded by a nucleotide sequence     having a cumulative human allele frequency from 1 to 10% and/or a     total human genotype frequency from 1 to about 15% or from 1 to 15%. -   46. The method, ligand, composition, kit or use of any preceding     paragraph wherein the TOI is a human TOI selected from Table 5;     optionally for treating and/or preventing a corresponding disease or     condition as set out in Table 5. -   47. A method of treating or preventing a disease or condition in a     human, wherein the disease or condition is mediated by a Target of     Interest (TOI), wherein the TOI is present in humans as different     polymorphic variants, the method comprising administering to the     human determined to be positive for the TOI polymorphic variant,     wherein the TOI in said human is encoded by a nucleotide sequence     comprising an allele having a cumulative human allele frequency of     less than 50% and/or wherein the TOI in said human is encoded by a     nucleotide sequence comprising an allele having total human genotype     frequency of less than 50% an anti-TOI ligand to target the TOI in     the human to treat or prevent said disease or condition. -   48. The method of paragraph 47, wherein the anti-TOI ligand is     selected from an antibody, an antibody portion, an antibody     fragment, an affibody, an antisense oligonucleotide, an siRNA, and a     microRNA. -   49. An anti-TOI ligand that specifically binds to a TOI as defined     in paragraph 1, for use in the method of any preceding paragraph     (eg, for use in the method of any of paragraphs 1 to 8 and 19 to     23). -   50. The ligand of paragraph 49, wherein the ligand is an anti-TOI     trap, antibody or antibody fragment. -   51. The ligand of paragraph 49 or 50, wherein the ligand is for     subcutaneous or intravenous administration to a human, or is     comprised by an injectable preparation. -   52. An injection device comprising the ligand of any one of     paragraphs 49 to 51.

Additional Tailoring of Ligands to Genotype and/or Phenotype of the Human Patient

As described herein, the present invention contemplates ligands (eg, antibodies and fragments) whose binding site specificities have been matched to one or more variant human TOIs (eg, PCSK9 or IL6R). Additionally or alternatively (and as further illustrated in the non-limiting Examples below), an optional aspect of the invention provides for matching of other features of the ligand to the patient's genotype or phenotype. In this respect, for example, the invention includes the ability to match amino acid sequence variation in a human patient to one or more ligand sequences or domains outside of the binding sites. For example, where the ligand comprises or consists of a human TOI-binding antibody or an anti-human TOI receptor Fc fusion, this aspect of the invention provides for more tailored matching of one or more constant region domains (eg, the Fc) to the patient genotype or phenotype. Additionally or alternatively, it is contemplated that sequence variation in the binding site can be similarly matched to the patient's genotype or phenotype. The present inventor has done this by considering the SNP occurrences in sequences encoding one or more parts of the ligand, eg, SNP occurrences in one, more or all of the gene segments from which the variable domain(s) and/or constant region domain(s) are derived. The inventor realised that it would be desirable to match the ligand to one or more corresponding variant SNPs found in the patient to be treated therapeutically and/or prophylactically. Matching could involve designing the ligand specifically for a patient of known phenotype and/or genotype, or matching could involve choosing a ligand by determining that there is correspondence between variation in the patient's phenotype or genotype with the variation in the ligand amino acid and/or corresponding nucleotides.

A key consideration for the inventor was the desire to promote compatibility of the ligand with the patient's body, and in particular, the possible patient immune system responses to administered ligands. For example, it has been observed that human patients receiving human or humanised antibody drugs may mount an immune response against the incoming antibody (a so-called HAHA response) which results in the patient producing anti-drug antibodies as a result of the patient's immune system recognising the drug as foreign. For example, studies have suggested that some patients receiving HUMIRA™ (adalimumab), currently the biggest selling antibody medicine, mount a HAHA immune response against the medicine, and this may impact treatment adversely. Reference is made to JAMA. 2011; 305(14):1460-1468. doi:10.1001/jama.2011.406: “Development of Antidrug Antibodies Against Adalimumab and Association With Disease Activity and Treatment Failure During Long-term Follow-up”, G M Bartelds et al. The authors concluded that results of this study showed that development of antidrug antibodies was associated with a negative outcome of adalimumab treatment in human RA patients. It was reported that not only did patients with anti-adalimumab antibodies discontinue treatment more often and earlier than patients without anti-adalimumab antibodies, they also had a higher disease activity during treatment and only rarely came into remission. In addition, reportedly the data showed that two-thirds of the anti-adalimumab antibody-positive patients developed these antibodies in the first 28 weeks of treatment and that the presence of anti-adalimumab antibodies substantially influenced serum adalimumab concentrations.

This HAHA theme is, therefore, a significant concern and this has been considered by the regulatory authorities. For example, the European Medicines Agency (EMA) has issued a “Guideline on immunogenicity assessment of monoclonal antibodies intended for in vivo clinical use” (available on the world wide web at ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/06/WC500128688.pdf; EMA/CHMP/BMWP/86289/2010, addendum to EMEA/CHMP/BMWP/14327/2006), which came into force on 1 Dec. 2012. As such, it is good practice for researchers to identify and assess risk of anti-antibody drug occurrence and effects.

The present aspect of the invention, by more closely tailoring the ligand itself (as well as its specificity) to the patient, helps to address these considerations when designing and administering anti-human TOI medicines for treating and/or preventing human TOI-related diseases and conditions.

The inventor also considered the desirability to tailor the variation in the ligand constant region (eg, for an antibody or Fc-containing ligand), mindful that then the constant region being administered to the patient would be tuned to the various components, such as patient's Fc receptors, that would interact with the constant region in the patient. Good Fc/Fc receptor interactions can be important for drug recycling (via the FcRn) to provide for useful half-lives in vivo or for use in cell killing, eg, for cancer indications. In this way it is possible, therefore, to tune the effector function of the constant region (eg, Fc) to the patient more closely, to promote efficacy. For example, more efficacious drugs are desirable for better patient treatment and may provide the possibility of lowered dosing and/or dosing frequencies.

Thus, in examples of this aspect, the invention provides the following (set out as clauses):—

-   1. The ligand, method, use, kit or composition of the invention,     wherein     -   (i) the ligand (eg, antibody or fragment) comprises         -   (c) a variable domain that is encoded by a human V region             nucleotide sequence, wherein the V nucleotide sequence is             derived from recombination of human VH, D and JH gene             segments or human VL and JL gene segments; or         -   (d) a constant region domain encoded by a C region gene             segment; Wherein a first gene segment of said gene segments             of (a), or said C region gene segment of         -   (b) comprises a first single nucleotide polymorphism (SNP)             encoding a first amino acid polymorphism; and     -   (ii) the genome of said human comprises said first SNP or         wherein said human expresses (a′) an antibody variable domain         comprising said first amino acid polymorphism or (b′) an         antibody constant domain comprising said first amino acid         polymorphism. -   2. The ligand, method, use, kit or composition of clause 1, wherein     blood of said human comprises substantially no antibodies that     specifically bind to the domain comprising said first amino acid     polymorphism as determined in an in vitro binding assay. -   3. The ligand, method, use, kit or composition of clause 2, wherein     SPR is used to carry out said assay.

In an alternative, ELISA is used.

-   4. The ligand, method, use, kit or composition of any one of clauses     1 to 3, wherein the genome of said human comprises said first gene     segment (when (a) applies) or said C region gene segment (when (b)     applies). -   5. The ligand, method, use, kit or composition of any one of clauses     1 to 4, wherein said first segment or a second segment of said     segments of (a), or said C region gene segment of (b), comprises a     second SNP encoding a second amino acid polymorphism; and wherein     the genome of said human comprises said second SNP or wherein said     human expresses (a″) an antibody variable domain comprising said     second amino acid polymorphism or (b″) an antibody constant region     domain comprising said first and second amino acid polymorphisms. -   6. The ligand, method, use, kit or composition of clause 5, wherein     said human expresses an antibody variable domain comprising said     first and second amino acid polymorphisms. -   7. The ligand, method, use, kit or composition of clause 5 or 6,     wherein the first and second SNPs of said genome are comprised by     the same antibody gene segment.

For example, the first and second SNPs of the genome are comprised by an IGHG1*01 gene segment and said first segment of (a) is an IGHG1*01 gene segment.

For example, the first and second SNPs of the genome are comprised by an IGHG2*01 gene segment and said first segment of (a) is an IGHG2*01 gene segment.

-   8. The ligand, method, use, kit or composition of any one of clauses     1 to 7, wherein each SNP is a variable region gene segment SNP. -   9. The ligand, method, use, kit or composition of any one of clauses     1 to 7, wherein each SNP is a constant region gene segment SNP, eg     each SNP is a gamma-1 constant region gene segment SNP, or a gamma-2     constant region gene segment SNP, or a gamma-3 constant region gene     segment SNP or a gamma-4 constant region gene segment SNP. -   10. The ligand, method, use, kit or composition of clause 9, wherein     the first SNP is a CH1, CH2, CH3 or CH4 gene segment SNP and/or the     second SNP is a CH1, CH2, CH3 or CH4 gene segment SNP. -   11. The ligand, method, use, kit or composition of any one of     clauses 1 to 8, wherein each SNP is a variable domain SNP, eg, a VH     domain SNP, or a Vκ domain SNP, or a Vλ SNP. -   12. The ligand, method, use, kit or composition of any one of     clauses 1 to 11, wherein said constant region domain of (b) is     comprised by an antibody Fc region. -   13. The ligand, method, use, kit or composition of any one of     clauses 1 to 12, wherein the ligand (eg, antibody or fragment) has     been determined to specifically bind one or more human TOI variants     as disclosed herein, for example, with a KD of 1 nM or less (eg, 100     or 10 pM or less) as determined by SPR. -   14. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 13), wherein the ligand     comprises or consists of an antibody or fragment that comprises a     human antibody variable domain derived from the recombination of a     human V gene segment and a human J gene segment (and optionally a     human D gene segment when the variable domains are VH domains); and     wherein the genome of the human comprises said human V gene segment     and/or the human expresses antibodies comprising antibody variable     domains derived from the recombination of said human V gene segment     and a human J gene segment (and optionally a human D gene segment).

In an example, the V gene segment is any of the V gene segments disclosed in WO2013041844, a 1000 Genomes database and/or www.imgt.org, the disclosures of which (including disclosure relating to sequence) is explicitly incorporated herein by reference for use in the present invention.

-   15. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 14), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused human TOI receptor) comprises a human heavy chain constant     domain encoded by a first constant region nucleotide sequence; and     wherein the genome of the human comprises a heavy chain constant     region nucleotide sequence that is identical to said first constant     region nucleotide sequence and/or the human expresses antibodies     comprising said human constant domain. -   16. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 15), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused human TOI receptor) comprises a human gamma heavy chain CH1     domain encoded by a CH1 nucleotide sequence; and wherein the genome     of the human comprises a gamma heavy chain constant region     nucleotide sequence that is identical to said CH1 nucleotide     sequence and/or the human expresses antibodies comprising said human     gamma CH1 domain. -   17. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 16), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused human TOI receptor) comprises a human gamma heavy chain CH2     domain encoded by a CH2 nucleotide sequence; and wherein the genome     of the human comprises a gamma heavy chain constant region     nucleotide sequence that is identical to said CH2 nucleotide     sequence and/or the human expresses antibodies comprising said human     gamma CH2 domain. -   18. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 17), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused human TOI receptor) comprises a human gamma heavy chain CH3     domain encoded by a CH3 nucleotide sequence; and wherein the genome     of the human comprises a gamma heavy chain constant region     nucleotide sequence that is identical to said CH3 nucleotide     sequence and/or the human expresses antibodies comprising said human     gamma CH3 domain. -   19. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 18), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused human TOI receptor) comprises a human gamma heavy chain CH4     domain encoded by a CH4 nucleotide sequence; and wherein the genome     of the human comprises a gamma heavy chain constant region     nucleotide sequence that is identical to said CH4 nucleotide     sequence and/or the human expresses antibodies comprising said human     gamma CH4 domain. -   20. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 19), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused human TOI receptor) comprises a human gamma heavy chain Fc     region encoded by a Fc nucleotide sequence; and wherein the genome     of the human comprises a gamma heavy chain constant region     nucleotide sequence that is identical to said Fc nucleotide sequence     and/or the human expresses antibodies comprising said human gamma Fc     region. -   21. The ligand, method, use, kit or composition of any one of     clauses 16 to 20, wherein said human gamma heavy chain is a human     gamma-1 heavy chain. -   22. The ligand, method, use, kit or composition of any one of     clauses 16 to 20, wherein said human gamma heavy chain is a human     gamma-2 heavy chain. -   23. The ligand, method, use, kit or composition of any one of     clauses 16 to 20, wherein ligand comprises a human IGHG1*01 gamma-1     heavy chain constant region. -   24. The ligand, method, use, kit or composition of any one of     clauses 16 to 20, wherein ligand comprises a human IGHG2*01 gamma-1     heavy chain constant region. -   25. The ligand, method, use, kit or composition of any one of     clauses 15 to 24, wherein the human has been or is genotyped as     positive for said heavy chain constant region nucleotide sequence. -   26. The ligand, method, use, kit or composition of clause 23,     wherein the human has been or is genotyped as positive for human     IGHG1*01 nucleotide sequence. -   27. The ligand, method, use, kit or composition of clause 24,     wherein the human has been or is genotyped as positive for human     IGHG2*01 nucleotide sequence. -   28. The ligand, method, use, kit or composition of any one of     clauses 16 to 24, wherein the human has been or is phenotyped as     positive for said gamma heavy chain constant domain, CH1, CH2, CH3,     CH4 or Fc. -   29. The ligand, method, use, kit or composition of clause 28, (i)     when dependent from clause 23, wherein the human has been or is     phenotyped as positive for a human IGHG1*01 gamma heavy chain     constant domain, CH1, CH2, CH3, CH4 or Fc or (ii) when dependent     from clause 24, wherein the human has been phenotyped as positive     for a human IGHG2*01 gamma heavy chain constant domain, CH1, CH2,     CH3, CH4 or Fc. -   30. The method or use of any one of clauses 16 to 24 and 26 to 29,     comprising genotyping the human as positive for said gamma heavy     chain constant region nucleotide sequence, eg, positive for said     gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc     nucleotide sequence; positive for said human IGHG1*01 gamma heavy     chain constant region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence;     or positive for said human IGHG2*01 gamma heavy chain constant     region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence. -   31. The method or use of any one of clauses 16 to 24 and 26 to 30,     comprising phenotyping the human as positive for said gamma heavy     chain constant region, eg, positive for said gamma heavy chain     constant domain, CH1, CH2, CH3, CH4 or Fc; positive for said human     IGHG1*01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or     Fc; or positive for said human IGHG2*01 gamma heavy chain constant     domain, CH1, CH2, CH3, CH4 or Fc.

Examples of Tailored Ligands

The inventor analysed amino acid variability and distribution amongst large representative human samples. The result of the analysis for example antibody gene segments is shown in Table 9.

In a first example, the inventor identified the possibility of addressing the rarer IGH-gamma-1 SNPs 204D (observed cumulative frequency of 0.296) and 206L (observed cumulative frequency of 0.283) individually or in combination. These residues are part of the CH3 domain, and as such they form part of antibody Fc regions. Thus, matching of these CH3 variations with the patient is especially beneficial for reasons as discussed above. Thus, this example provides aspects set out in the following clauses.

-   32. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 31), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused TOI receptor) comprises a human gamma-1 heavy chain     constant region that comprises an Asp corresponding to position 204     of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID     NO: 42 and wherein the genome of the human comprises a gamma-1 heavy     chain constant region nucleotide sequence that encodes such an Asp     or Leu or the human expresses antibodies comprising human gamma-1     constant regions comprising such an Asp or Leu.

The skilled person will be familiar with techniques for determining genome sequences of a human, eg, by using a sample containing genomic DNA and/or RNA, sequencing and comparing using bioinformatics or other computer tools to compare the sampled sequence with sequences of human alleles (eg, as shown in the IMGT, 100 genomes or other database as disclosed herein). In an example, the sample is a blood or saliva or cheek swab sample.

-   33. The ligand, method, use, kit or composition of clause 32,     wherein the ligand comprises a human gamma-1 heavy chain constant     region that comprises an Asp corresponding to position 204 of SEQ ID     NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42. -   34. The ligand, method, use, kit or composition of clause 32 or 33,     wherein the genome of the human comprises a gamma-1 heavy chain     constant region nucleotide sequence that encodes such an Asp and Leu     or the human expresses antibodies comprising human gamma-1 constant     regions comprising such an Asp and Leu. -   35. The ligand, method, use, kit or composition of clause 32, 33 or     34, wherein the ligand comprises a human IGHG1*01 gamma-1 heavy     chain constant region, eg, an Fc, CH1, CH2 and/or CH3 domain encoded     by human IGHG1*01. -   36. The ligand, method, use, kit or composition of any one of     clauses 32 to 35, wherein the genome of the human comprises a human     IGHG1*01 nucleotide sequence or the human expresses antibodies     comprising human constant domains encoded by a human IGHG1*01     nucleotide sequence. -   37. The ligand, method, use, kit or composition of any one of     clauses 32 to 36, wherein the ligand comprises a hinge region     encoded by human IGHG1*01. -   38. The ligand, method, use, kit or composition of any one of     clauses 32 to 37, wherein the ligand comprises or consists of an     antibody, wherein the antibody comprises heavy chains that comprise     SEQ ID NO: 61. -   39. The ligand, method, use, kit or composition of any one of     clauses 32 to 38, wherein the human is of European ancestry.

As shown in Table 9, 204D and 206L are found in such humans.

-   40. The ligand, method, use, kit or composition of any one of     clauses 32 to 39, wherein the human has been or is genotyped as     positive for said Asp and/or Leu. -   41. The ligand, method, use, kit or composition of any one of     clauses 32 to 40, wherein the human has been or is genotyped as     positive for human IGHG1*01. -   42. The ligand, method, use, kit or composition of any one of     clauses 32 to 41, wherein the human has been or is phenotyped as     positive for a human IGHG1*01 CH3. -   43. The method or use of any one of clauses 32 to 42, comprising     selecting a said human whose genome comprises a codon(s) encoding     said Asp and/or Leu; comprises human IGHG1*01; or comprises a human     IGHG1*01 CH3. -   44. The method or use of any one of clauses 32 to 43, comprising     selecting a said human whose phenotype comprises said Asp and/or     Leu; a human IGHG1*01 region; or a human IGHG1*01 CH3.     -   44a. The ligand, method, use, kit or composition of any one of         clauses 32 to 44, wherein the human expresses antibodies         comprising human gamma-1 constant regions comprising such an Asp         and Leu.

In a second example, the inventor identified the possibility of addressing IGH-gamma-2 SNPs. This included consideration of Fc region variation—in this respect, the inventor focused on positions 161 and 257 which are in the Fc region. Thus, this example provides aspects set out in the following clauses.

-   45. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 31), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused TOI receptor) comprises a human gamma-2 heavy chain     constant region that comprises an amino acid selected from the group     consisting of a Pro corresponding to position 72 of SEQ ID NO: 44,     an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe     corresponding to position 76 of SEQ ID NO: 44, a Val corresponding     to position 161 of SEQ ID NO: 44 and an Ala corresponding to     position 257 of SEQ ID NO: 44; and wherein the genome of the human     comprises a gamma-2 heavy chain constant region nucleotide sequence     that encodes such a selected amino acid or the human expresses     antibodies comprising human gamma-2 constant regions comprising such     a selected amino acid. -   46. The ligand, method, use, kit or composition of clause 45,     wherein the ligand comprises a human gamma-2 heavy chain constant     region that comprises (i) a Pro corresponding to position 72 of SEQ     ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a     Phe corresponding to position 76 of SEQ ID NO: 44 and     optionally (ii) a Val corresponding to position 161 of SEQ ID NO: 44     and/or an Ala corresponding to position 257 of SEQ ID NO: 44; and     wherein the genome of the human comprises a gamma-2 heavy chain     constant region nucleotide sequence that encodes such amino acids     of (i) or the human expresses antibodies comprising human gamma-2     constant regions comprising such amino acids of (i).

This example focuses on CH1 variation.

-   47. The ligand, method, use, kit or composition of clause 45 or 46,     wherein the ligand comprises a human gamma-2 heavy chain constant     region that comprises (i) a Val corresponding to position 161 of SEQ     ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44     and optionally (ii) an amino acid selected from the group consisting     of a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn     corresponding to position 75 of SEQ ID NO: 44 and a Phe     corresponding to position 76 of SEQ ID NO: 44; and wherein the     genome of the human comprises a gamma-2 heavy chain constant region     nucleotide sequence that encodes such amino acids of (i) or the     human expresses antibodies comprising human gamma-2 constant regions     comprising such amino acids of (i).

This example focuses on Fc variation.

-   48. The ligand, method, use, kit or composition of any one of     clauses 45 to 47, wherein the ligand comprises a human IGHG2*01     gamma-2 heavy chain constant region, eg, an Fc, CH1, CH2 and/or CH3     domain encoded by human IGHG2*01. -   49. The ligand, method, use, kit or composition of any one of     clauses 45 to 48, wherein the genome of the human comprises a human     IGHG2*01 nucleotide sequence or the human expresses antibodies     comprising human constant domains encoded by a human IGHG2*01     nucleotide sequence. -   50. The ligand, method, use, kit or composition of any one of     clauses 45 to 49, wherein the ligand comprises a hinge region     encoded by human IGHG2*01. -   51. The ligand, method, use, kit or composition of any one of     clauses 45 to 50, wherein the ligand comprises or consists of an     antibody, wherein the antibody comprises heavy chains that comprise     SEQ ID NO: 63 or 65. -   52. The ligand, method, use, kit or composition of any one of     clauses 45 to 51, wherein the human is of European, African     American, or European American ancestry. -   53. The ligand, method, use, kit or composition of any one of     clauses 45 to 52, wherein the human has been or is genotyped as     positive for one, more or all of said Pro, Asn, Phe, Val and Ala. -   54. The ligand, method, use, kit or composition of any one of     clauses 45 to 53, wherein the human has been or is genotyped as     positive for human IGHG2*01. -   55. The ligand, method, use, kit or composition of any one of     clauses 45 to 54, wherein the human has been or is phenotyped as     positive for a human IGHG2*01 CH1. -   56. The ligand, method, use, kit or composition of any one of     clauses 45 to 55, wherein the human has been or is phenotyped as     positive for a human IGHG2*01 CH2. -   57. The ligand, method, use, kit or composition of any one of     clauses 45 to 56, wherein the human has been or is phenotyped as     positive for a human IGHG2*01 CH3. -   58. The method or use of any one of clauses 45 to 57, comprising     selecting a said human whose genome comprises a codon(s) encoding     one, more or all of said Pro, Asn, Phe, Val and Ala; comprises human     IGHG2*01; or comprises a human IGHG2*01 CH1, CH2 and/or CH3. -   59. The method or use of any one of clauses 45 to 58, comprising     selecting a said human whose phenotype comprises one, more or all of     said Pro, Asn, Phe, Val and Ala; a human IGHG2*01 region; or a human     IGHG2*01 CH1, CH2 and/or CH3. -   60. The ligand, method, use, kit or composition of any one of     clauses 45 to 59, wherein the human expresses antibodies comprising     human gamma-2 constant regions comprising such a Pro, Asn, Phe, Val     and Ala.

In a third example, the inventor addressed human kappa constant region variation. Thus, the present aspect of the invention also provides the following.

-   61. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 60), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused TOI receptor) comprises a human kappa light chain constant     region that comprises a Val corresponding to position 84 of SEQ ID     NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50; and     wherein the genome of the human comprises a kappa light chain     constant region nucleotide sequence that encodes such a Val or Cys     or the human expresses antibodies comprising human kappa light chain     constant regions comprising such a Val or Cys. -   62. The ligand, method, use, kit or composition of clause 61,     wherein the ligand comprises a human kappa light chain constant     region that comprises a Val corresponding to position 84 of SEQ ID     NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50. -   63. The ligand, method, use, kit or composition of clause 61 or 62,     wherein the genome of the human comprises a kappa light chain     constant region nucleotide sequence that encodes such a Val and Cys     or the human expresses antibodies comprising human kappa constant     regions comprising such a Val and Cys. -   64. The ligand, method, use, kit or composition of any one of     clauses 61 to 63, wherein the antibody or fragment comprises a human     IGKC*01 kappa light chain constant region. -   65. The ligand, method, use, kit or composition of any one of     clauses 61 to 64, wherein the ligand comprises or consists of an     antibody, wherein the antibody comprises light chains that comprise     SEQ ID NO: 62 or 66. -   66. The ligand, method, use, kit or composition of any one of     clauses 61 to 65, wherein the ligand comprises or consists of an     antibody, wherein the antibody comprises a light chain variable     domain derived from recombination of a human Vκ gene segment and a     human Jκ gene segment, wherein the Jκ gene segment is IGKJ2*01 (SEQ     ID NO: 57). -   67. The ligand, method, use, kit or composition of any one of     clauses 61 to 66, wherein the human has been or is phenotyped as     positive for said Val and/or Cys. -   68. The ligand, method, use, kit or composition of any one of     clauses 61 to 67, wherein the human has been or is genotyped as     positive for human IGKC*01. -   69. The ligand, method, use, kit or composition of any one of     clauses 61 to 68, wherein the human has been or is phenotyped as     positive for a human IGKC*01 domain. -   70. The method or use of any one of clauses 61 to 69, comprising     selecting a said human whose genome comprises a codon(s) encoding     said Val and/or Cys; or comprises human IGKC*01. -   71. The method or use of any one of clauses 61 to 70, comprising     selecting a said human whose phenotype comprises such a Val and/or     Cys; or comprises a human IGKC*01 domain. -   72. The ligand, method, use, kit or composition of any one of     clauses 61 to 71, wherein the human expresses antibodies comprising     human kappa constant domains comprising such a Val and Cys, eg,     expresses human IGKC*01 constant domains.

In a fourth example, the inventor addressed human lambda constant region variation. Thus, this example provides aspects set out in the following clauses.

-   73. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 60), wherein the ligand     (eg, comprising or consisting of an antibody or fragment or an     Fc-fused TOI receptor) comprises a human IGLC2*01 light chain     constant region; and wherein the genome of the human comprises a     human IGLC2*01 nucleotide sequence or the human expresses antibodies     comprising human light chain IGLC2*01 constant regions. -   74. The ligand, method, use, kit or composition of clause 73,     wherein the antibody comprises light chains that comprise SEQ ID NO:     64. -   75. The ligand, method, use, kit or composition of clause 73 or 74,     wherein the human has been or is genotyped as positive for human     IGLC2*01. -   76. The ligand, method, use, kit or composition of any one of     clauses 73 to 75, wherein the human has been or is phenotyped as     positive for a human IGLC2*01 domain. -   77. The method or use of any one of clauses 73 to 76, comprising     selecting a said human whose genome comprises human IGLC2*01. -   78. The method or use of any one of clauses 73 to 77, comprising     selecting a said human whose phenotype comprises a human IGLC2*01     domain. -   79. The ligand, method, use, kit or composition of any one of     clauses 73 to 78, wherein the human expresses antibodies comprising     human lambda IGLC2*01 constant domains.

In a fifth example, the inventor addressed human heavy chain variable region variation. Thus, this example provides aspects set out in the following clauses.

-   80. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 79), wherein the ligand     comprises or consists of an antibody or fragment, wherein the     antibody or fragment comprises a VH domain that is derived from the     recombination of a human VH gene segment, a human D gene segment and     a human JH gene segment, wherein the VH gene segment is selected     from the group consisting of (i) IGHV1-18*01 and the genome of the     human comprises a human IGHV1-18*01 nucleotide sequence or the human     expresses antibodies comprising variable domains derived from the     recombination of human IGHV1-18*01; or (ii) IGVH1-46*01 and the     genome of the human comprises a human IGHV1-46*01 nucleotide     sequence or the human expresses antibodies comprising variable     domains derived from the recombination of human IGHV1-46*01. -   81. The ligand, method, use, kit or composition of clause 80,     wherein the antibody or fragment comprises a one more or all of a     CH1 domain, CH2 domain, CH3 domain, hinge or Fc encoded by human     IGHG2*01. -   82. The ligand, method, use, kit or composition of clause 80 or 81,     wherein the antibody or fragment comprises heavy chains that     comprise SEQ ID NO: 63 or 65. -   83. The ligand, method, use, kit or composition of any one of     clauses 80 to 82, wherein the human has been or is genotyped as     positive for said selected VH gene segment, positive for human     IGHV1-18*01 or IGVH1-46*01. -   84. The method or use of any of clauses 80 to 83, comprising     genotyping the human as positive for said selected VH gene segment,     eg, positive for human IGHV1-18*01 or IGVH1-46*01.

In a sixth example, the inventor addressed human light chain variable region variation. Thus, this example provides aspects set out in the following clauses.

-   85. The ligand, method, use, kit or composition of the invention     (eg, according to any one of clauses 1 to 84), wherein the ligand     comprises or consists of an antibody or fragment, wherein the     antibody or fragment comprises a VL domain that is derived from the     recombination of a human VL gene segment and a human JL gene     segment, wherein the VL gene segment is selected from the group     consisting of (i) IGKV4-1*01 and the genome of the human comprises a     human IGKV4-1*01 nucleotide sequence or the human expresses     antibodies comprising variable domains derived from the     recombination of human IGKV4-1*01; (ii) IGLV2-14*01 and the genome     of the human comprises a human IGLV2-14*01 nucleotide sequence or     the human expresses antibodies comprising variable domains derived     from the recombination of human IGLV2-14*01; or (iii) IGKV1-13*02     and the genome of the human comprises a human IGKV1-13*02 nucleotide     sequence or the human expresses antibodies comprising variable     domains derived from the recombination of human IGKV1-13*02. -   86. The ligand, method, use, kit or composition of clause 85,     wherein the antibody comprises light chains that comprise SEQ ID NO:     62, 64 or 66. -   87. The ligand, method, use, kit or composition of clause 85 or 86,     wherein the antibody or fragment comprises a light chain variable     domain derived from recombination of a human Vκ gene segment and a     human Jκ gene segment, wherein the Jκ gene segment is IGKJ2*01 (SEQ     ID NO: 57; wherein (i) or (iii) applies. -   88. The ligand, method, use, kit or composition of any one of     clauses 85 to 87, wherein the human has been or is genotyped as     positive for said selected VL gene segment, eg, positive for human     IGKV4-1*01, IGLV2-14*01 or IGKV1-13*02. -   89. The method or use of clause 88, comprising genotyping the human     as positive for said selected VL gene segment, eg, genotyping the     human as positive for human IGKV4-1*01, IGLV2-14*01 or IGKV1-13*02. -   90. The ligand, method, use, kit or composition of any one of     clauses 1 to 89, wherein the ligand (eg, -   antibody or fragment) binds said human TOI with a dissociation     constant (Kd) of 1 nM or less as determined by SPR, (eg, 100, 10 or     1 pM or less).

In a specific embodiment, the ligand, antibody or fragment of present invention comprises an Fc region, wherein the Fc region comprises at least one non-native amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may comprise additional and/or alternative non-native amino acid residues known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

In an example the ligand, antibody or fragment comprises one or more human binding sites that specifically bind the TOI (eg, PCSK9, IL4Ra, IL6R, VEGFA or Nav1.7). In an embodiment, the binding sites comprise or consist of human antibody variable domains or human receptors for the TOI (eg, Human receptor binding sites for VEGFA). Furthermore, the ligand, antibody or fragment can be optionally fully human (eg, comprising human constant regions, eg, human Fc regions with or without human CL regions). In an example, the ligand is aflibercept, alirocumab, sarilumab or dupilumab. Fully human ligands maximise compatibility with the human patient when used in the context of the invention wherein the V and/or C regions are tailored to the human genotype and/or phenotype as per the description herein.

Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

-   1. A method of treating or preventing a disease or condition in a     human, wherein the disease or condition is mediated by a Target of     Interest (TOI), wherein the TOI is present in humans as different     polymorphic variants, the method comprising     -   a. Administering to the human an anti-TOI ligand to target a TOI         variant in the human and treat or prevent said disease or         condition, wherein the TOI in said human is encoded by a         nucleotide sequence having a cumulative human allele frequency         of less than 50% and/or wherein the TOI in said human is encoded         by a nucleotide sequence having a total human genotype frequency         of less than 50%;         -   wherein     -   b. Before step (a) said human has been or is genotyped as         positive for said nucleotide sequence or phenotyped as positive         for said TOI variant. -   2. The method of paragraph 1, wherein before step (a) the ligand has     been or is determined as being capable of binding to said TOI     variant. -   3. A method of treating or preventing a disease or condition in a     human, wherein the disease or condition is mediated by a Target of     Interest (TOI), wherein the TOI is present in humans as different     polymorphic variants, the method comprising     -   a. Administering to the human an anti-TOI ligand to target a TOI         variant in the human and treat or prevent said disease or         condition, wherein the TOI in said human is encoded by a         nucleotide sequence having a cumulative human allele frequency         of less than 50% and/or wherein the TOI in said human is encoded         by a nucleotide sequence having a total human genotype frequency         of less than 50%;         -   wherein     -   b. Before step (a) the ligand has been or is determined as         capable of binding to said TOI variant. -   4. The method of paragraph 3, wherein the genome of said human     comprises a nucleotide sequence encoding said TOI variant; and     before step (a) said nucleotide sequence has been or is determined     as having a cumulative human allele frequency of less than 50%     and/or having a total human genotype frequency of less than 50%. -   5. The method of paragraph 3 or 4, wherein said human has been or is     genotyped as positive for said variant nucleotide sequence before     step (a). -   6. The method of any preceding paragraph, wherein the human has been     or is phenotyped as positive for said TOI variant before step (a). -   7. The method of any preceding paragraph, wherein said frequency is     less than 10 or 15%. -   8. The method of any preceding paragraph, wherein the ligand is     capable of binding to two or more different TOI variants, each being     encoded by a nucleotide sequence having a cumulative human allele     frequency of less than 50% and/or having a total human genotype     frequency of less than 50%. -   9. A method of treating or preventing a disease or condition in a     human, wherein the disease or condition is mediated by a Target of     Interest (TOI), wherein the TOI is present in humans as different     polymorphic variants, the method comprising     -   a. Administering to the human an anti-TOI ligand to target a TOI         variant in the human and treat or prevent said disease or         condition, wherein the TOI in said human is a variant encoded by         a nucleotide sequence having a cumulative human allele frequency         of more than 50% and/or having a total human genotype frequency         of more than 50%;         -   wherein     -   b. Before step (a) said human has been or is genotyped as         negative for a variant nucleotide sequence having a cumulative         human allele frequency of less than 50% and/or having a total         human genotype frequency of less than 50%; or phenotyped as         negative for a TOI variant encoded by a nucleotide sequence         having a cumulative human allele frequency of less than 50%         and/or having a total human genotype frequency of less than 50%. -   10. The method of paragraph 9, wherein before step (a), the human     has been or is phenotyped as positive for the most frequent TOI     variant or genotyped for the nucleotide sequence thereof. -   11. The method of paragraph 9 or 10, wherein before step (a) the     ligand has been or is determined as being capable of binding to the     most frequent TOI variant. -   12. The method of paragraph 9, 10 or 11, wherein before step (a) the     ligand has been or is determined as being substantially incapable of     neutralising or inhibiting said TOI variant recited in step (b). -   13. The method of any one of paragraphs 9 to 12, wherein the ligand     is capable of binding to the most frequent TOI variant. -   14. The method of any one of paragraphs 9 to 13, wherein the ligand     is capable of binding to two or more different TOI variants, each     being encoded by a nucleotide sequence having a cumulative human     allele frequency of more than 50%. -   15. The method of any preceding paragraph, wherein said variant     nucleotide sequence recited in step (a) has been or is determined as     being present in at least 2 different human ethnic populations. -   16. The method of any preceding paragraph, wherein said human     frequency is the frequency in a database of naturally-occurring     sequences sampled from at least 15 different human ethnic     populations and comprising at least 1000 sequences. -   17. An anti-human TOI ligand for use in a method of treating and/or     preventing a TOI-mediated disease or condition in a human, wherein     the TOI is present in humans as different polymorphic variants and     wherein the genome of said human comprises a TOI nucleotide sequence     having a cumulative human allele frequency of less than 50% and/or a     total human genotype frequency of less than 50%, the method     comprising administering the ligand to the human. -   18. The ligand of paragraph 17, wherein the ligand has been or is     determined as capable of binding the human TOI encoded by said     nucleotide sequence. -   19. A ligand that binds a human TOI comprising an amino acid     sequence encoded by a TOI nucleotide sequence having a cumulative     human allele frequency of less than 50% and/or a total human     genotype frequency of less than 50%, for use in a method comprising     the step of using the ligand to target said TOI in a human to treat     and/or prevent a disease or condition mediated by TOI, the method     comprising administering the ligand to the human. -   20. The ligand of any one of paragraphs 17 to 19, wherein the human     has been or is genotyped as positive for said TOI nucleotide     sequence having a cumulative human allele frequency of less than     50%.

The ligand of any one of paragraphs 17 to 19, wherein the human has been or is genotyped as positive for said TOI nucleotide sequence having a total human genotype frequency of less than 50%.

-   21. The ligand of any one of paragraphs 17 to 20, wherein the human     has been or is phenotyped as positive for a TOI encoded by a     nucleotide sequence having a cumulative human allele frequency of     less than 50% and/or having a total human genotype frequency of less     than 50%. -   22. The ligand of any one of paragraphs 17 to 21, wherein the human     has been or is genotyped as heterozygous for a TOI nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or having a total human genotype frequency of less than 50%;     optionally wherein the human has been or is genotyped as comprising     a TOI nucleotide sequence having a cumulative human allele frequency     of less than 50% and a TOI nucleotide sequence having a cumulative     human allele frequency of more than 50% and/or having a total human     genotype frequency of more than 50%. -   23. The ligand of any one of paragraphs 17 to 22, wherein the genome     of the human has been or is genotyped as homozygous for a TOI     nucleotide sequence having a cumulative human allele frequency of     less than 50% and/or having a total human genotype frequency of less     than 50%. -   24. The ligand of any one of paragraphs 17 to 23, wherein the ligand     comprises an antibody binding site that binds a human TOI comprising     an amino acid sequence encoded by a TOI nucleotide sequence having a     cumulative human allele frequency of less than 50% and/or having a     total human genotype frequency of less than 50%; and optionally has     been or is determined as capable of such binding. -   25. The ligand of paragraph 24, wherein the ligand is an antibody or     antibody fragment. -   26. The ligand of any one of paragraphs 17 to 23, wherein the ligand     comprises a nucleotide sequence that specifically hybridises to a     TOI nucleotide sequence having a cumulative human allele frequency     of less than 50% and/or having a total human genotype frequency of     less than 50% or an RNA transcript thereof; and/or the ligand     comprises a nucleotide sequence that comprises at least 10     contiguous nucleotides of a nucleotide sequence having a cumulative     human allele frequency of less than 50% and/or having a total human     genotype frequency of less than 50% or is an antisense sequence     thereof. -   27. The ligand of any one of paragraphs 17 to 26, wherein the genome     of said human comprises a nucleotide sequence having a cumulative     human allele frequency of less than 50% and the sequence is found in     at least 2 different ethnic populations. -   28. A pharmaceutical composition or kit for treating or preventing a     condition or disease mediated by a TOI as recited in any preceding     paragraph, the composition or kit comprising a ligand of any one of     paragraphs 17 to 27; and optionally in combination with a label or     instructions for use to treat and/or prevent said disease or     condition in a human; optionally wherein the label or instructions     comprise a marketing authorisation number (eg, an FDA or EMA     authorisation number); optionally wherein the kit comprises an     injection pen or IV container that comprises the ligand. -   29. A method of producing an anti-human TOI antibody binding site,     the method comprising obtaining a plurality of anti-TOI antibody     binding sites, screening the antibody binding sites for binding to a     TOI comprising an amino acid sequence encoded by a nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or a total human genotype frequency of less than 50%, or to a     peptide thereof that comprises an amino acid variation from the     corresponding sequence encoded by the TOI-encoding nucleotide     sequence having the highest cumulative human allele frequency and/or     the highest total human genotype frequency, and isolating an     antibody binding site that binds in the screening step. -   30. A method of producing an anti-human TOI antibody, the method     comprising immunising a non-human vertebrate (eg, a mouse or a rat)     with a TOI comprising an amino acid sequence encoded by a nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or a total human genotype frequency of less than 50%, or to a     peptide thereof that comprises an amino acid variation from the     corresponding sequence encoded by the TOI-encoding nucleotide     sequence having the highest cumulative human allele frequency and/or     the highest total human genotype frequency, and isolating an     antibody that binds a TOI comprising an amino acid sequence encoded     by a TOI nucleotide sequence having a cumulative human allele     frequency of less than 50% and/or a total human genotype frequency     of less than 50%, and optionally producing a TOI-binding fragment or     derivative of the isolated antibody. -   31. The method of paragraph 29 or 30, comprising the step of     obtaining a nucleic acid encoding the antibody, fragment, derivative     or binding site and optionally inserting the nucleic acid in an     expression vector. -   32. A kit for TOI genotyping a human, wherein the kit comprises a     nucleic acid comprising a nucleotide sequence that specifically     hybridises to a TOI nucleotide sequence selected having a cumulative     human allele frequency of less than 50% and/or a total human     genotype frequency of less than 50% or an RNA transcript thereof;     and/or the nucleic acid comprises a nucleotide sequence that     comprises at least 10 contiguous nucleotides of a TOI nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or a total human genotype frequency of less than 50% or is an     antisense sequence thereof. -   33. A kit for TOI genotyping or phenotyping a human, wherein the kit     comprises a ligand according to any one of paragraphs 17 to 27 or an     antibody, fragment or derivative produced by the method of any one     of paragraphs 29 to 31. -   34. Use of an anti-TOI ligand that binds a human TOI comprising an     amino acid sequence encoded by a TOI nucleotide sequence having a     cumulative human allele frequency of less than 50% and/or a total     human genotype frequency of less than 50%, in the manufacture of a     medicament for treating and/or preventing a TOI-mediated disease or     condition in a human whose genome comprises a TOI nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or having a total human genotype frequency of less than 50%. -   35. Use of an anti-TOI ligand that binds a human TOI comprising an     amino acid sequence encoded by a TOI nucleotide sequence having a     cumulative human allele frequency of less than 50% and/or a total     human genotype frequency of less than 50%, in the manufacture of a     medicament for targeting said TOI in a human to treat and/or prevent     a disease or condition mediated by TOI. -   36. A method of targeting a TOI for treating and/or preventing a     TOI-mediated disease or condition in a human, the method comprising     administering an anti-TOI ligand to a human comprising a TOI     nucleotide sequence selected having a cumulative human allele     frequency of less than 50% and/or a total human genotype frequency     of less than 50%, whereby a TOI encoded by said nucleotide sequence     is targeted. -   37. The method of paragraph 36, wherein the method comprises     targeting a human TOI comprising an amino acid sequence with said     ligand to treat and/or prevent said disease or condition in said     human, wherein said amino acid sequence is encoded by a nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or a total human genotype frequency of less than 50%. -   38. A method of TOI genotyping a nucleic acid sample of a human, the     method comprising identifying in the sample the presence of a TOI     nucleotide sequence having a cumulative human allele frequency of     less than 50% and/or having a total human genotype frequency of less     than 50%. -   39. A method of TOI typing a protein sample of a human, the method     comprising identifying in the sample the presence of a TOI amino     acid sequence encoded by a TOI nucleotide sequence having a     cumulative human allele frequency of less than 50% and/or having a     total human genotype frequency of less than 50%. -   40. The method of paragraph 38 or 39, comprising obtaining a sample     of serum, blood, faeces, hair, urine or saliva from a human, whereby     the nucleic acid or protein sample is obtained for use in the step     of identifying said sequence. -   41. The method of any one of paragraphs 38 to 40, comprising using a     ligand according to any one of paragraphs 17 to 27 to carry out said     identifying step. -   42. A diagnostic kit comprising a ligand that is capable of binding     a human TOI comprising an amino acid sequence encoded by a TOI     nucleotide sequence having a cumulative human allele frequency of     less than 50% and/or a total human genotype frequency of less than     50% and instructions for carrying out the method of paragraph 38 or     39. -   43. A diagnostic kit comprising a nucleic acid probe comprising a     nucleotide sequence that specifically hybridises a TOI nucleotide     sequence having a cumulative human allele frequency of less than 50%     and/or a total human genotype frequency of less than 50% or an RNA     transcript thereof and instructions for carrying out the method of     paragraph 38 or 39. -   44. The method, ligand, composition, kit or use of any preceding     paragraph, wherein the TOI is encoded by a nucleotide sequence     having a cumulative human allele frequency from 1 to 10% and/or a     total human genotype frequency from 1 to about 15% or from 1 to 15%. -   45. The method, ligand, composition, kit or use of any preceding     paragraph wherein the TOI is a human TOI selected from Table 5;     optionally for treating and/or preventing a corresponding disease or     condition as set out in table 5. -   46. The ligand, method, use, kit or composition of any preceding     paragraph, wherein     -   (i) the ligand (eg, antibody or fragment) comprises         -   (e) a variable domain that is encoded by a human V region             nucleotide sequence, wherein the V nucleotide sequence is             derived from recombination of human VH, D and JH gene             segments or human VL and JL gene segments; or         -   (f) a constant region domain encoded by a C region gene             segment; Wherein a first gene segment of said gene segments             of (a), or said C region gene segment of (b) comprises a             first single nucleotide polymorphism (SNP) encoding a first             amino acid polymorphism; and     -   (ii) the genome of said human comprises said first SNP or         wherein said human expresses (a′) an antibody variable domain         comprising said first amino acid polymorphism or (b′) an         antibody constant domain comprising said first amino acid         polymorphism. -   47. The ligand, method, use, kit or composition of paragraph 46,     wherein blood of said human comprises substantially no antibodies     that specifically bind to the domain comprising said first amino     acid polymorphism as determined in an in vitro binding assay. -   48. The ligand, method, use, kit or composition of paragraph 47,     wherein SPR is used to carry out said assay. -   49. The ligand, method, use, kit or composition of any one of     paragraphs 46 to 48, wherein the genome of said human comprises said     first gene segment (when (a) applies) or said C region gene segment     (when (b) applies). -   50. The ligand, method, use, kit or composition of any one of     paragraphs 46 to 49, wherein said first segment or a second segment     of said segments of (a), or said C region gene segment of (b),     comprises a second SNP encoding a second amino acid polymorphism;     and wherein the genome of said human comprises said second SNP or     wherein said human expresses (a″) an antibody variable domain     comprising said second amino acid polymorphism or (b″) an antibody     constant region domain comprising said first and second amino acid     polymorphisms. -   51. The ligand, method, use, kit or composition of paragraph 50,     wherein said human expresses an antibody variable domain comprising     said first and second amino acid polymorphisms. -   52. The ligand, method, use, kit or composition of paragraph 50 or     51, wherein the first and second SNPs of said genome are comprised     by the same antibody gene segment. -   53. The ligand, method, use, kit or composition of any one of     paragraphs 46 to 52, wherein each SNP is a variable region gene     segment SNP. -   54. The ligand, method, use, kit or composition of any one of     paragraphs 46 to 52, wherein each SNP is a constant region gene     segment SNP, eg each SNP is a gamma-1 constant region gene segment     SNP, or a gamma-2 constant region gene segment SNP, or a gamma-3     constant region gene segment SNP or a gamma-4 constant region gene     segment SNP. -   55. The ligand, method, use, kit or composition of paragraph 54,     wherein the first SNP is a CH1, CH2, CH3 or CH4 gene segment SNP     and/or the second SNP is a CH1, CH2, CH3 or CH4 gene segment SNP. -   56. The ligand, method, use, kit or composition of any one of     paragraphs 46 to 53, wherein each SNP is a variable domain SNP, eg,     a VH domain SNP, or a Vκ domain SNP, or a VW SNP. -   57. The ligand, method, use, kit or composition of any one of     paragraphs 46 to 56, wherein said constant region domain of (b) is     comprised by an antibody Fc region. -   58. The ligand, method, use, kit or composition of any one of     paragraphs 46 to 57, wherein the ligand (eg, antibody or fragment)     has been determined to specifically bind one or more human TOI     variants as disclosed herein, for example, with a KD of 1 nM or less     (eg, 100 or 10 pM or less) as determined by SPR. -   59. The ligand, method, use, kit or composition of any preceding     paragraph (eg, according to any one of paragraphs 46 to 58), wherein     the ligand comprises or consists of an antibody or fragment that     comprises a human antibody variable domain derived from the     recombination of a human V gene segment and a human J gene segment     (and optionally a human D gene segment when the variable domains are     VH domains); and wherein the genome of the human comprises said     human V gene segment and/or the human expresses antibodies     comprising antibody variable domains derived from the recombination     of said human V gene segment and a human J gene segment (and     optionally a human D gene segment). -   60. The ligand, method, use, kit or composition of any preceding     paragraph (eg, according to any one of paragraphs 46 to 59), wherein     the ligand (eg, comprising or consisting of an antibody or fragment     or an Fc-fused human TOI receptor) comprises a human heavy chain     constant domain encoded by a first constant region nucleotide     sequence; and wherein the genome of the human comprises a heavy     chain constant region nucleotide sequence that is identical to said     first constant region nucleotide sequence and/or the human expresses     antibodies comprising said human constant domain. -   61. The ligand, method, use, kit or composition of any preceding     paragraph (eg, according to any one of paragraphs 46 to 60), wherein     the ligand (eg, comprising or consisting of an antibody or fragment     or an Fc-fused human TOI receptor) comprises a human gamma heavy     chain CH1 domain encoded by a CH1 nucleotide sequence; and wherein     the genome of the human comprises a gamma heavy chain constant     region nucleotide sequence that is identical to said CH1 nucleotide     sequence and/or the human expresses antibodies comprising said human     gamma CH1 domain. -   62. The ligand, method, use, kit or composition of any preceding     paragraph (eg, according to any one of paragraphs 46 to 61), wherein     the ligand (eg, comprising or consisting of an antibody or fragment     or an Fc-fused human TOI receptor) comprises a human gamma heavy     chain CH2 domain encoded by a CH2 nucleotide sequence; and wherein     the genome of the human comprises a gamma heavy chain constant     region nucleotide sequence that is identical to said CH2 nucleotide     sequence and/or the human expresses antibodies comprising said human     gamma CH2 domain. -   63. The ligand, method, use, kit or composition of any preceding     paragraph (eg, according to any one of paragraphs 46 to 62),     wherein, wherein the ligand (eg, comprising or consisting of an     antibody or fragment or an Fc-fused human TOI receptor) comprises a     human gamma heavy chain CH3 domain encoded by a CH3 nucleotide     sequence; and wherein the genome of the human comprises a gamma     heavy chain constant region nucleotide sequence that is identical to     said CH3 nucleotide sequence and/or the human expresses antibodies     comprising said human gamma CH3 domain. -   64. The ligand, method, use, kit or composition of any preceding     paragraph (eg, according to any one of paragraphs 46 to 63), wherein     the ligand (eg, comprising or consisting of an antibody or fragment     or an Fc-fused human TOI receptor) comprises a human gamma heavy     chain CH4 domain encoded by a CH4 nucleotide sequence; and wherein     the genome of the human comprises a gamma heavy chain constant     region nucleotide sequence that is identical to said CH4 nucleotide     sequence and/or the human expresses antibodies comprising said human     gamma CH4 domain. -   65. The ligand, method, use, kit or composition of any preceding     paragraph (eg, according to any one of paragraphs 46 to 64), wherein     the ligand (eg, comprising or consisting of an antibody or fragment     or an Fc-fused human TOI receptor) comprises a human gamma heavy     chain Fc region encoded by a Fc nucleotide sequence; and wherein the     genome of the human comprises a gamma heavy chain constant region     nucleotide sequence that is identical to said Fc nucleotide sequence     and/or the human expresses antibodies comprising said human gamma Fc     region. -   66. The ligand, method, use, kit or composition of any one of     paragraphs 61 to 65, wherein said human gamma heavy chain is a human     gamma-1 heavy chain. -   67. The ligand, method, use, kit or composition of any one of     paragraphs 61 to 65, wherein said human gamma heavy chain is a human     gamma-2 heavy chain. -   68. The ligand, method, use, kit or composition of any one of     paragraphs 61 to 65, wherein ligand comprises a human IGHG1*01     gamma-1 heavy chain constant region. -   69. The ligand, method, use, kit or composition of any one of     paragraphs 61 to 65, wherein ligand comprises a human IGHG2*01     gamma-1 heavy chain constant region. -   70. The ligand, method, use, kit or composition of any one of     paragraphs 60 to 69, wherein the human has been or is genotyped as     positive for said heavy chain constant region nucleotide sequence. -   71. The ligand, method, use, kit or composition of paragraph 68,     wherein the human has been or is genotyped as positive for human     IGHG1*01 nucleotide sequence. -   72. The ligand, method, use, kit or composition of paragraph 69,     wherein the human has been or is genotyped as positive for human     IGHG2*01 nucleotide sequence. -   73. The ligand, method, use, kit or composition of any one of     paragraphs 61 to 69, wherein the human has been or is phenotyped as     positive for said gamma heavy chain constant domain, CH1, CH2, CH3,     CH4 or Fc. -   74. The ligand, method, use, kit or composition of paragraph 63, (i)     when dependent from clause 23, wherein the human has been or is     phenotyped as positive for a human IGHG1*01 gamma heavy chain     constant domain, CH1, CH2, CH3, CH4 or Fc or (ii) when dependent     from clause 24, wherein the human has been phenotyped as positive     for a human IGHG2*01 gamma heavy chain constant domain, CH1, CH2,     CH3, CH4 or Fc. -   75. The method or use of any one of paragraphs 61 to 69 and 71 to     74, comprising genotyping the human as positive for said gamma heavy     chain constant region nucleotide sequence, eg, positive for said     gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc     nucleotide sequence; positive for said human IGHG1*01 gamma heavy     chain constant region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence;     or positive for said human IGHG2*01 gamma heavy chain constant     region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence. -   76. The method or use of any one of paragraphs 61 to 69 and 71 to     75, comprising phenotyping the human as positive for said gamma     heavy chain constant region, eg, positive for said gamma heavy chain     constant domain, CH1, CH2, CH3, CH4 or Fc; positive for said human     IGHG1*01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or     Fc; or positive for said human IGHG2*01 gamma heavy chain constant     domain, CH1, CH2, CH3, CH4 or Fc. -   77. The ligand, method, use, kit or composition of any preceding     paragraph (eg, according to any one of clauses 46 to 76), wherein     the ligand (eg, comprising or consisting of an antibody or fragment     or an Fc-fused TOI receptor) comprises a human gamma-1 heavy chain     constant region that comprises an Asp corresponding to position 204     of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID     NO: 42 and wherein the genome of the human comprises a gamma-1 heavy     chain constant region nucleotide sequence that encodes such an Asp     or Leu or the human expresses antibodies comprising human gamma-1     constant regions comprising such an Asp or Leu. -   78. The ligand, method, use, kit or composition of paragraph 77,     wherein the ligand comprises a human gamma-1 heavy chain constant     region that comprises an Asp corresponding to position 204 of SEQ ID     NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42. -   79. The ligand, method, use, kit or composition of paragraph 77 or     78, wherein the genome of the human comprises a gamma-1 heavy chain     constant region nucleotide sequence that encodes such an Asp and Len     or the human expresses antibodies comprising human gamma-1 constant     regions comprising such an Asp and Leu. -   80. The ligand, method, use, kit or composition of paragraph 77, 78     or 79, wherein the ligand comprises a human IGHG1*01 gamma-1 heavy     chain constant region, eg, an Fc, CH1, CH2 and/or CH3 domain encoded     by human IGHG1*01. -   81. The ligand, method, use, kit or composition of any one of     paragraphs 77 to 80, wherein the genome of the human comprises a     human IGHG1*01 nucleotide sequence or the human expresses antibodies     comprising human constant domains encoded by a human IGHG1*01     nucleotide sequence. -   82. The ligand, method, use, kit or composition of any one of     paragraphs 77 to 81, wherein the ligand comprises a hinge region     encoded by human IGHG1*01. -   83. The ligand, method, use, kit or composition of any one of     paragraphs 77 to 82, wherein the ligand comprises or consists of an     antibody, wherein the antibody comprises heavy chains that comprise     SEQ ID NO: 61. -   84. The ligand, method, use, kit or composition of any one of     paragraphs 77 to 83, wherein the human is of European ancestry. -   85. The ligand, method, use, kit or composition of any one of     paragraphs 77 to 84, wherein the human has been or is genotyped as     positive for said Asp and/or Leu. -   86. The ligand, method, use, kit or composition of any one of     paragraphs 77 to 85, wherein the human has been or is genotyped as     positive for human IGHG1*01. -   87. The ligand, method, use, kit or composition of any one of     paragraphs 77 to 86, wherein the human has been or is phenotyped as     positive for a human IGHG1*01 CH3. -   88. The method or use of any one of paragraphs 77 to 87, comprising     selecting a said human whose genome comprises a codon(s) encoding     said Asp and/or Leu; comprises human IGHG1*01; or comprises a human     IGHG1*01 CH3. -   89. The method or use of any one of paragraphs 77 to 88, comprising     selecting a said human whose phenotype comprises said Asp and/or     Leu; a human IGHG1*01 region; or a human IGHG1*01 CH3. -   90. The ligand, method, use, kit or composition of any preceding     paragraph, wherein the ligand (eg, comprising or consisting of an     antibody or fragment or an Fc-fused TOI receptor) comprises a human     gamma-2 heavy chain constant region that comprises an amino acid     selected from the group consisting of a Pro corresponding to     position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of     SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44,     a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala     corresponding to position 257 of SEQ ID NO: 44; and wherein the     genome of the human comprises a gamma-2 heavy chain constant region     nucleotide sequence that encodes such a selected amino acid or the     human expresses antibodies comprising human gamma-2 constant regions     comprising such a selected amino acid. -   91. The ligand, method, use, kit or composition of paragraph 90,     wherein the ligand comprises a human gamma-2 heavy chain constant     region that comprises (i) a Pro corresponding to position 72 of SEQ     ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a     Phe corresponding to position 76 of SEQ ID NO: 44 and     optionally (ii) a Val corresponding to position 161 of SEQ ID NO: 44     and/or an Ala corresponding to position 257 of SEQ ID NO: 44; and     wherein the genome of the human comprises a gamma-2 heavy chain     constant region nucleotide sequence that encodes such amino acids     of (i) or the human expresses antibodies comprising human gamma-2     constant regions comprising such amino acids of (i). -   92. The ligand, method, use, kit or composition of paragraph 90 or     91, wherein the ligand comprises a human gamma-2 heavy chain     constant region that comprises (i) a Val corresponding to position     161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ     ID NO: 44 and optionally (ii) an amino acid selected from the group     consisting of a Pro corresponding to position 72 of SEQ ID NO: 44,     an Asn corresponding to position 75 of SEQ ID NO: 44 and a Phe     corresponding to position 76 of SEQ ID NO: 44; and wherein the     genome of the human comprises a gamma-2 heavy chain constant region     nucleotide sequence that encodes such amino acids of (i) or the     human expresses antibodies comprising human gamma-2 constant regions     comprising such amino acids of (i). -   93. The ligand, method, use, kit or composition of any one of     paragraph 90 to 92, wherein the ligand comprises a human IGHG2*01     gamma-2 heavy chain constant region, eg, an Fc, CH1, CH2 and/or CH3     domain encoded by human IGHG2*01. -   94. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 93, wherein the genome of the human comprises a     human IGHG2*01 nucleotide sequence or the human expresses antibodies     comprising human constant domains encoded by a human IGHG2*01     nucleotide sequence. -   95. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 94, wherein the ligand comprises a hinge region     encoded by human IGHG2*01. -   96. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 95, wherein the ligand comprises or consists of an     antibody, wherein the antibody comprises heavy chains that comprise     SEQ ID NO: 63 or 65. -   97. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 96, wherein the human is of European, African     American, or European American ancestry. -   98. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 97, wherein the human has been or is genotyped as     positive for one, more or all of said Pro, Asn, Phe, Val and Ala. -   99. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 98, wherein the human has been or is genotyped as     positive for human IGHG2*01. -   100. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 99, wherein the human has been or is phenotyped as     positive for a human IGHG2*01 CH1. -   101. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 100, wherein the human has been or is phenotyped as     positive for a human IGHG2*01 CH2. -   102. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 101, wherein the human has been or is phenotyped as     positive for a human IGHG2*01 CH3. -   103. The method or use of any one of paragraphs 90 to 102,     comprising selecting a said human whose genome comprises a codon(s)     encoding one, more or all of said Pro, Asn, Phe, Val and Ala;     comprises human IGHG2*01; or comprises a human IGHG2*01 CH1, CH2     and/or CH3. -   104. The method or use of any one of paragraphs 90 to 103,     comprising selecting a said human whose phenotype comprises one,     more or all of said Pro, Asn, Phe, Val and Ala; a human IGHG2*01     region; or a human IGHG2*01 CH1, CH2 and/or CH3. -   105. The ligand, method, use, kit or composition of any one of     paragraphs 90 to 104, wherein the human expresses antibodies     comprising human gamma-2 constant regions comprising such a Pro,     Asn, Phe, Val and Ala. -   106. The ligand, method, use, kit or composition of any preceding     paragraph, wherein the ligand (eg, comprising or consisting of an     antibody or fragment or an Fc-fused TOI receptor) comprises a human     kappa light chain constant region that comprises a Val corresponding     to position 84 of SEQ ID NO: 50 or a Cys corresponding to position     87 of SEQ ID NO: 50; and wherein the genome of the human comprises a     kappa light chain constant region nucleotide sequence that encodes     such a Val or Cys or the human expresses antibodies comprising human     kappa light chain constant regions comprising such a Val or Cys. -   107. The ligand, method, use, kit or composition of paragraph 106,     wherein the ligand comprises a human kappa light chain constant     region that comprises a Val corresponding to position 84 of SEQ ID     NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50. -   108. The ligand, method, use, kit or composition of paragraph 106 or     107, wherein the genome of the human comprises a kappa light chain     constant region nucleotide sequence that encodes such a Val and Cys     or the human expresses antibodies comprising human kappa constant     regions comprising such a Val and Cys. -   109. The ligand, method, use, kit or composition of any one of     paragraphs 106 to 108, wherein the antibody or fragment comprises a     human IGKC*01 kappa light chain constant region. -   110. The ligand, method, use, kit or composition of any one of     paragraphs 106 to 109, wherein the ligand comprises or consists of     an antibody, wherein the antibody comprises light chains that     comprise SEQ ID NO: 62 or 66. -   111. The ligand, method, use, kit or composition of any one of     paragraphs 106 to 110, wherein the ligand comprises or consists of     an antibody, wherein the antibody comprises a light chain variable     domain derived from recombination of a human Vκ gene segment and a     human Jκ gene segment, wherein the Jκ gene segment is IGKJ2*01 (SEQ     ID NO: 57). -   112. The ligand, method, use, kit or composition of any one of     paragraphs 106 to 111, wherein the human has been or is phenotyped     as positive for said Val and/or Cys. -   113. The ligand, method, use, kit or composition of any one of     paragraphs 106 to 112, wherein the human has been or is genotyped as     positive for human IGKC*01. -   114. The ligand, method, use, kit or composition of any one of     paragraphs 106 to 113, wherein the human has been or is phenotyped     as positive for a human IGKC*01 domain. -   115. The method or use of any one of paragraphs 106 to 114,     comprising selecting a said human whose genome comprises a codon(s)     encoding said Val and/or Cys; or comprises human IGKC*01. -   116. The method or use of any one of paragraphs 106 to 115,     comprising selecting a said human whose phenotype comprises such a     Val and/or Cys; or comprises a human IGKC*01 domain. -   117. The ligand, method, use, kit or composition of any one of     paragraphs 106 to 116, wherein the human expresses antibodies     comprising human kappa constant domains comprising such a Val and     Cys, eg, expresses human IGKC*01 constant domains. -   118. The ligand, method, use, kit or composition of any preceding     paragraph, wherein the ligand (eg, comprising or consisting of an     antibody or fragment or an Fc-fused TOI receptor) comprises a human     IGLC2*01 light chain constant region; and wherein the genome of the     human comprises a human IGLC2*01 nucleotide sequence or the human     expresses antibodies comprising human light chain IGLC2*01 constant     regions. -   119. The ligand, method, use, kit or composition of paragraph 118,     wherein the antibody comprises light chains that comprise SEQ ID NO:     64. -   120. The ligand, method, use, kit or composition of paragraph 118 or     119, wherein the human has been or is genotyped as positive for     human IGLC2*01. -   121. The ligand, method, use, kit or composition of any one of     paragraphs 118 to 120, wherein the human has been or is phenotyped     as positive for a human IGLC2*01 domain. -   122. The method or use of any one of paragraphs 118 to 212,     comprising selecting a said human whose genome comprises human     IGLC2*01. -   123. The method or use of any one of clauses 73 to 77, comprising     selecting a said human whose phenotype comprises a human IGLC2*01     domain. -   124. The ligand, method, use, kit or composition of any one of     paragraphs 108 to 123, wherein the human expresses antibodies     comprising human lambda IGLC2*01 constant domains. -   125. The ligand, method, use, kit or composition of any preceding     paragraph, wherein the ligand comprises or consists of an antibody     or fragment, wherein the antibody or fragment comprises a VH domain     that is derived from the recombination of a human VH gene segment, a     human D gene segment and a human JH gene segment, wherein the VH     gene segment is selected from the group consisting of (i)     IGHV1-18*01 and the genome of the human comprises a human     IGHV1-18*01 nucleotide sequence or the human expresses antibodies     comprising variable domains derived from the recombination of human     IGHV1-18*01; or (ii) IGVH1-46*01 and the genome of the human     comprises a human IGHV1-46*01 nucleotide sequence or the human     expresses antibodies comprising variable domains derived from the     recombination of human IGHV1-46*01. -   126. The ligand, method, use, kit or composition of paragraph 125,     wherein the antibody or fragment comprises a one more or all of a     CH1 domain, CH2 domain, CH3 domain, hinge or Fc encoded by human     IGHG2*01. -   127. The ligand, method, use, kit or composition of paragraph 125 or     126, wherein the antibody or fragment comprises heavy chains that     comprise SEQ ID NO: 63 or 65. -   128. The ligand, method, use, kit or composition of any one of     paragraphs 125 to 127, wherein the human has been or is genotyped as     positive for said selected VH gene segment, positive for human     IGHV1-18*01 or IGVH1-46*01. -   129. The method or use of any of paragraphs 125 to 128, comprising     genotyping the human as positive for said selected VH gene segment,     eg, positive for human IGHV1-18*01 or IGVH1-46*01. -   130. The ligand, method, use, kit or composition any preceding     paragraph, wherein the ligand comprises or consists of an antibody     or fragment, wherein the antibody or fragment comprises a VL domain     that is derived from the recombination of a human VL gene segment     and a human JL gene segment, wherein the VL gene segment is selected     from the group consisting of (i) IGKV4-1*01 and the genome of the     human comprises a human IGKV4-1*01 nucleotide sequence or the human     expresses antibodies comprising variable domains derived from the     recombination of human IGKV4-1*01; (ii) IGLV2-14*01 and the genome     of the human comprises a human IGLV2-14*01 nucleotide sequence or     the human expresses antibodies comprising variable domains derived     from the recombination of human IGLV2-14*01; or (iii) IGKV1-13*02     and the genome of the human comprises a human IGKV1-13*02 nucleotide     sequence or the human expresses antibodies comprising variable     domains derived from the recombination of human IGKV1-13*02. -   131. The ligand, method, use, kit or composition of paragraph 130,     wherein the antibody comprises light chains that comprise SEQ ID NO:     62, 64 or 66. -   132. The ligand, method, use, kit or composition of paragraph 130 or     131, wherein the antibody or fragment comprises a light chain     variable domain derived from recombination of a human Vκ gene     segment and a human Jκ gene segment, wherein the Jκ gene segment is     IGKJ2*01 (SEQ ID NO: 57; wherein (i) or (iii) applies. -   133. The ligand, method, use, kit or composition of any one of     paragraphs 130 to 132, wherein the human has been or is genotyped as     positive for said selected VL gene segment, eg, positive for human     IGKV4-1*01, IGLV2-14*01 or IGKV1-13*02. -   134. The method or use of paragraph 133, comprising genotyping the     human as positive for said selected VL gene segment, eg, genotyping     the human as positive for human IGKV4-1*01, IGLV2-14*01 or     IGKV1-13*02. -   135. The ligand, method, use, kit or composition of any preceding     paragraph, wherein the ligand (eg, antibody or fragment) binds said     human TOI with a dissociation constant (Kd) of 1 nM or less as     determined by SPR, (eg, 100, 10 or 1 pM or less). -   136. The ligand, method, use, kit or composition of any preceding     paragraph, wherein the TOI is human PCSK9 or human IL-6R.

EXAMPLES Example 1: Rare PCSK9 Variants

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serine protease involved in regulating the levels of the low density lipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah and Prat, 2007). In vitro experiments have shown that adding PCSK9 to HepG2 cells lowers the levels of cell surface LDLR (Benjannet et al., 2004; Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004). Experiments with mice have shown that increasing PCSK9 protein levels decreases levels of LDLR protein in the liver (Benjannet et al., 2004; Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004), while PCSK9 knockout mice have increased levels of LDLR in the liver (Rashid et al., 2005). Additionally, various human PCSK9 mutations that result in either increased or decreased levels of plasma LDL have been identified (Kotowski et al., 2006; Zhao et al., 2006). PCSK9 has been shown to directly interact with the LDLR protein, be endocytosed along with the LDLR, and co-immunofluoresce with the LDLR throughout the endosomal pathway (Lagace et al., 2006).

PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 2003). Humans have nine prohormone-proprotein convertases that can be divided between the S8A and S8B subfamilies (Rawlings et al., 2006). Furin, PC1/PC3, PC2, PACE4, PC4, PC5/PC6 and PC7/PC8/LPC/SPC7 are classified in subfamily S8B. Crystal and NMR structures of different domains from mouse furin and PC1 reveal subtilisin-like pro- and catalytic domains, and a P domain directly C-terminal to the catalytic domain (Henrich et al., 2003; Tangrea et al., 2002). Based on the amino acid sequence similarity within this subfamily, all seven members are predicted to have similar structures (Henrich et al., 2005). SKI-1/SIP and PCSK9 are classified in subfamily S8A. Sequence comparisons with these proteins also suggest the presence of subtilisin-like pro- and catalytic domains (Sakai et al., 1998; Seidah et al., 2003; Seidah et al., 1999). In these proteins the amino acid sequence C-terminal to the catalytic domain is more variable and does not suggest the presence of a P domain.

Prohormone-proprotein convertases are expressed as zymogens and they mature through a multi step process. The function of the pro-domain in this process is two-fold. The pro-domain first acts as a chaperone and is required for proper folding of the catalytic domain (Ikemura et al., 1987). Once the catalytic domain is folded, autocatalysis occurs between the pro-domain and catalytic domain. Following this initial cleavage reaction, the pro-domain remains bound to the catalytic domain where it then acts as an inhibitor of catalytic activity (Fu et al., 2000). When conditions are correct, maturation proceeds with a second autocatalytic event at a site within the pro-domain (Anderson et al., 1997). After this second cleavage event occurs the pro-domain and catalytic domain dissociate, giving rise to an active protease.

Autocatalysis of the PCSK9 zymogen occurs between Gln152 and Ser153 (VFAQlSIP (SEQ ID NO: 116)) (Naureckiene et al., 2003), and has been shown to be required for its secretion from cells (Seidah et al., 2003). A second autocatalytic event at a site within PCSK9's pro-domain has not been observed. Purified PCSK9 is made up of two species that can be separated by non-reducing SDS-PAGE; the pro-domain at 17 Kd, and the catalytic plus C-terminal domains at 65 Kd. PCSK9 has not been isolated without its inhibitory pro-domain, and measurements of PCSK9's catalytic activity have been variable (Naureckiene et al., 2003; Seidah et al., 2003).

In certain embodiments, a PCSK9 polypeptide includes terminal residues, such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues. “PCSK9” has also been referred to as FH3, NARC1, HCHOLA3, proprotein convertase subtilisin/kexin type 9, and neural apoptosis regulated convertase 1. The PCSK9 gene encodes a proprotein convertase protein that belongs to the proteinase K subfamily of the secretory subtilase family. The term “PCSK9” denotes both the proprotein and the product generated following autocatalysis of the proprotein. When only the autocatalyzed product is being referred to (such as for an antigen binding protein or ligand that binds to the cleaved PCSK9), the protein can be referred to as the “mature,” “cleaved”, “processed” or “active” PCSK9. When only the inactive form is being referred to, the protein can be referred to as the “inactive”, “pro-form”, or “unprocessed” form of PCSK9. The term PCSK9 also encompasses PCSK9 molecules incorporating post-translational modifications of the PCSK9 amino acid sequence, such as PCSK9 sequences that have been glycosylated, PCSK9 sequences from which its signal sequence has been cleaved, PCSK9 sequence from which its pro domain has been cleaved from the catalytic domain but not separated from the catalytic domain (see, e.g., FIGS. 1A and 1B of US20120093818A1; which is incorporated by reference herein in its entirety).

The present invention provides anti-PCSK9 ligands; and PCSK9-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of PCSK9, in particular human PCSK9 or its ligands and in screening assays to identify other antagonists of PCSK9 activity. Some of the ligands of the invention are useful for inhibiting binding of PCSK9 to LDLR, or inhibiting PCSK9-mediated activities.

Anti-PCSK9 ligands (eg, antibodies and anti-sense RNA) have been developed based on targeting and neutralising so-called “wild-type” human PCSK9, which is a commonly-occurring form (see, eg, US20120093818A1 and US20110065902A1; each of which is incorporated by reference herein in its entirety). While such therapies are useful for human patients harbouring this form of human PCSK9, the inventor considered it useful to investigate the possibility of targeting much rarer—but still naturally-occurring—forms of PCSK9 amongst human populations. In this way, the inventor arrived at insight into the natural occurrences and distributions of rarer human PCSK9 forms that can serve as useful targets (at the protein or nucleic acid level) for human treatment, prophylaxis and diagnosis pertinent to diseases and conditions mediated or associated with PCSK9 activity. This particularly provides for tailored therapies, prophylaxis and diagnosis in humans that are devoid of the common PCSK9 gene or protein (ie, the form a or a′ as used in US20120093818A1 and US20110065902A1 to generate antibodies).

The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in activity and/or conformation of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to more effectively tailor medicines and diagnosis of patients. The invention, therefore, provides for tailored pharmaceuticals and testing that specifically addresses rarer PCSK9 polymorphic variant forms. Such forms or “alleles” (at the nucleotide level), in many of the examples determined by the inventor, comprise multiple changes at the nucleotide and amino acid levels from the corresponding common form nucleotide and amino acids sequences, ie, there are multiple non-synonymous changes at the nucleotide level that translate into multiple corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting such rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention.

With this realisation, the inventor realised that there is significant industrial and medical application for the invention in terms of guiding the choice of anti-PCSK9 ligand for administration to human patients for therapy and/or prophylaxis of PCSK9-mediated or associated diseases or conditions. In this way, the patient receives drugs and ligands that are tailored to their needs—as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

In developing this thinking, the present inventor decided to determine a set of human PCSK9 variants on the basis of the following criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population. The inventor selected variants having at least 3 of the 4 following criteria:—

-   -   PCSK9 variants having a cumulative human allele frequency in the         range from 1 to 10%;     -   PCSK9 variants having a total human genotype frequency in the         range from 1 to about 15%;     -   PCSK9 variants found in many different human ethnic populations         (using the standard categorisation of the 1000 Genomes Project,         which is an accepted standard in the art; see Table 4 below);         and     -   PCSK9 variants found in many individuals distributed across such         many different ethnic populations.

On the basis of these criteria, the inventor identified the variants listed in Table 1 below (excluding form a).

The inventor's selection included, as a consideration, selection for nucleotide variation that produced amino acid variation in corresponding PCSK9 forms (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.

TABLE 1 Human PCSK9 variants distributed over several human ethnic populations & having a total human genotype frequency in the range of 1 to about 15% (a) Amino acid variability, population distributions and frequencies ASW, YRI, GBR, TSI, CLM, CHB, LWK, CHS, MXL, 0.4506 Form a JPT, PUR, IBS, FIN, (0.8457) 46R 53A 425N 443A 474I 619Q 670E CEU 939 14 Hom Freq⁴ Variant Amino Acid Position & Variation Human No. No. Unique 0.3951 (Het + Hom 0.64815 Form 46L 53V 425S 443T 474V 619P 670G Populations Individs¹ Pops² Het Freq³ freq⁵) Cum Freq⁶ f x ASW, YRI, GBR, 180 12 0.153 0.009 0.0855 TSI, CLM, LWK, (0.162) MXL, JPT, PUR, IBS, FIN, CEU c x ASW, YRI, GBR, 153 12 0.1296 0.0081 0.0729 TSI, CLM, CHB, (0.1377) LWK, CHS, JPT, PUR, FIN, CEU r x x 0.0234 0.009 0.0292 (0.0324) p x x ASW, GBR, TSI, 49 9 0.0441 (0.0441) 0.0221 CLM, JPT, PUR, IBS, FIN, CEU m x LWK, ASW, YRI, 29 4 0.0225 (0.0225) 0.0149 CLM e x x LWK, ASW, YRI 15 3 0.0135 (0.0135) 0.0068 h x x LWK, ASW, YRI 10 3 0.009 (0.009) 0.0045 aj X x PUR, TSI, FIN, CEU 9 4 0.0081 (0.0081) 0.0041 q x x CHS, ASW, JPT, 7 5 0.0063 (0.0063) 0.0032 PUR, CHB Table Footnotes: “x” in a box indicates that the amino acid for the variant form is different from the amino acid at that position in form a, the variant amino acid being shown in “Amino Acid Position & Variation” of the table and the form a amino acid being shown in the first row of the table; amino acids at all other positions of each variant form are identical to those found in form a. Amino acid numbering is per the numbering shown for the pro-form in Table 2 below. ¹Number of individuals in 1000 Genomes database found to have the allele; ²Number of unique human ethnic populations in 1000 Genomes database in which the allele was found to occur; ³Heterozygous human genotype frequency, ie, cumulative frequency of all genotypes having one occurrence of the variant allele and one occurrence of another allele (heterozygous state), eg, ac genotype in 1000 Genomes database; ⁴Homozygous human genotype frequency, ie, cumulative frequency of two occurrences of the variant allele (homozygous state), eg, cc genotype in 1000 Genomes database; and ⁵Total human genotype frequency, ie, total of heterozygous plus homozygous human genotype frequencies. ⁶Cumulative human allele frequency of all occurrences of the variant allele in 1000 Genomes database. Form a′ is identical to form a with the exception that form a′ has a glycine (G) at position 620 (see US20120093818 (Amgen, Inc)); form a has E at this position. (b) Nucleotide Sequence Variations of Selected Alleles Allele a G C A G A A A Nucleotide Position¹ 1: 55505647 1: 55505668 1: 55523802 1: 55523855 1: 55524237 1: 55527222 1: 55529187 Non-Synonymous Nucleotide Variation² T T G A G C G Variant ID³ rs11591147 rs11583680 rs28362261 rs28362263 rs562556 rs28362277 rs505151 Corresponding Amino Acid Variation Variant Allele 46L 53V 425S 443T 474V 619P 670G f X c X r X X p X X m X e X X h X X aj X X q X X “x” in a box indicates that a variant allele comprises the non-synonymous nucleotide variation indicated in the 5th row. Table Footnotes: ¹Notation is chromosome number (all positions are on human chromosome 1): coordinate number (Ensembl release 73 - September 2013, Genome assembly: GRCh37 (GCA_000001405.13); ²Nucleotide change (compared to allele a nucleotide shown in first row) giving rise to an amino acid change in the variant form (compared to amino acid of allele a); and ³NCBI dbSNP reference number (NCBI dbSNP Build 138 released on Apr. 25, 2013).

TABLE 2 Sequences (a) Human PCSK9 Form a Amino Acid Sequence (SEQ ID NO: 1)-“Pro-form” with Signal Sequence                                             46     53 MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTA RRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIIDSSVFAQsipwnleritppryradeyqppdggslvevylldtsiqs dhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvl lplaggyrrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtigtnfgrcvdlfapgediigassdcstcfvs                                           425               443                           474 qsgtsqaahvagiaammlsaepeltlaelrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARC APDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLG                                            619 620 THKPPVLRPGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTS  670 EEAVTAVAICCRSRHLAQASQELQ Italics = signal sequence 1-30 Courier = pro peptide 31-152 lower case = catalytic domain 153-449 UPPER CASE = C-terminal domain 450-692 Underlined = residues changed from allele a in other sequences (aa residue number shown) The pro-form is the sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1. The mature form in the sequence from amino acid number 153 to (and including) amino acid number 692 of SEQ ID NO: 1. (b) Human PCSK9 form a Amino Acid Sequence (SEQ ID NO: 3)-“Mature-form” (Numbering and notation as per SEQ ID NO: 1 above has been retained) sipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasm rslvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqd                                                                                  425 qpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsakdivneawfpedqrvltp    443                            474 nlvaalppsghGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQAN                                                                                    619 620 CSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEE                                         670 GWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ (c) Human PCSK9 Allele a Nucleotide Sequence (SEQ ID NO: 28)-Encoding “Pro-form” Plus Signal Sequence ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGA                                      R46L CGT to CTT        A53V GCC to GTC GGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCT TCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCC CGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAG TGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctgg agcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacag agtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaa gtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctca actgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtg ctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaa cttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctgg ggactttggggaccaactttggccgctgtgtggacctctttgccccgaggggaggacatcattggtgcctccagcgactgcagcacctgctttgt gtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttga                                   N425S AAT to AGT                                    A443T GCC ggcagagactgatccacttctctgccaaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaacctggtggccgcc to ACC                                                                                I474V ATC ctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCCACAGCCATCGC to GTC CCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGC TGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACA GCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGA CCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCC                                      Q619P CAG to CCG E620G GAG to GGG ATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCAGGAGGGCTGGACCCTGACTG GCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGC         E670G GAG to GGG AGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC Italics = nucleotide sequence encoding signal sequence (nucleotides 1-90) Courier = nucleotide sequence encoding pro peptide (nucleotides 91-456) lower case = nucleotide sequence encoding catalytic domain (nucleotides 457-1346) UPPER CASE = nucleotide sequence encoding C-terminal domain (nucleotides 1347-2076) Underlined = allelic variations from allele a in other sequences (aa residue number changes and codon changes shown) The pro-form is encoded by nucleotide sequence from nucleotide 91 to (and including) nucleotide 2076. The mature form is encoded by nucleotide sequence from nucleotide 457 to (and including) nucleotide 2076.

Variant Allele Nucleotide Sequences

Thus,

(i) The nucleotide sequence of allele f is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele f comprises a GTC codon instead of an ATC codon at the position labelled “I474V” in SEQ ID NO: 28; (ii) The nucleotide sequence of allele c is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele c comprises a GGG codon instead of an GAG codon at the position labelled “E670G” in SEQ ID NO: 28; (iii) The nucleotide sequence of allele r is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele r comprises a GTC codon instead of an ATC codon at the position labelled “I474V” in SEQ ID NO: 28; and a GGG codon instead of an GAG codon at the position labelled “E670G” in SEQ ID NO: 28; (iv) The nucleotide sequence of allele p is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele p comprises a GTC codon instead of a GCC codon at the position labelled “A53V” in SEQ ID NO: 28; and a GTC codon instead of an ATC codon at the position labelled “I474V” in SEQ ID NO: 28; (v) The nucleotide sequence of allele m is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele m comprises a ACC codon instead of a GCC codon at the position labelled “A443T” in SEQ ID NO: 28; (vi) The nucleotide sequence of allele e is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele e comprises a AGT codon instead of an AAT codon at the position labelled “N425S” in SEQ ID NO: 28; and a GTC codon instead of an ATC codon at the position labelled “I474V” in SEQ ID NO: 28; (vii) The nucleotide sequence of allele h is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele h comprises a ACC codon instead of a GCC codon at the position labelled “A443T” in SEQ ID NO: 28; and a CCG codon instead of a CAG codon at the position labelled “Q619P” in SEQ ID NO: 28; (viii) The nucleotide sequence of allele aj is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele aj comprises a CTT codon instead of an CGT codon at the position labelled “R46L” in SEQ ID NO: 28; and a GTC codon instead of an ATC codon at the position labelled “I474V” in SEQ ID NO: 28; and (ix) The nucleotide sequence of allele q is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele q comprises a GTC codon instead of a GCC codon at the position labelled “A53V” in SEQ ID NO: 28; and a GGG codon instead of an GAG codon at the position labelled “E670G” in SEQ ID NO: 28.

Variant Pro-Form Amino Acid Sequences

(Numbering is as per SEQ ID NO: 1 recited above)

(A) The amino acid sequence of form f is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form f comprises a valine at position 474; (B) The amino acid sequence of form c is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form c comprises a glycine at position 670; (C) The amino acid sequence of form r is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form r comprises a valine at position 474 and a glycine at position 670; (D) The amino acid sequence of form p is identical the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form p comprises a valine at position 53 and a valine at position 474; (E) The amino acid sequence of form m is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form m comprises a threonine at position 443; (F) The amino acid sequence of form e is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form e comprises a serine at position 425 and a valine at position 474; (G) The amino acid sequence of form h is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form h comprises a threonine at position 443 and a proline at position 619; (H) The amino acid sequence of form aj is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form aj comprises a leucine at position 46 and a valine at position 474; and (I) The amino acid sequence of form q is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form q comprises a valine at position 53 and a glycine at position 670.

Variant Mature Form Amino Acid Sequences

(Numbering is as per SEQ ID NO: 1 recited above)

(A′) The amino acid sequence of form f is identical to SEQ ID NO: 2 except that the amino acid sequence of form f comprises a valine at position 474; (B′) The amino acid sequence of form c is identical to SEQ ID NO: 2 except that the amino acid sequence of form c comprises a glycine at position 670; (C′) The amino acid sequence of form r is identical to SEQ ID NO: 2 except that the amino acid sequence of form r comprises a valine at position 474 and a glycine at position 670; (D′) The amino acid sequence of form p is identical to SEQ ID NO: 2 except that the amino acid sequence of form p comprises a valine at position 474; (E′) The amino acid sequence of form m is identical to SEQ ID NO: 2 except that the amino acid sequence of form m comprises a threonine at position 443; (F′) The amino acid sequence of form e is identical to SEQ ID NO: 2 except that the amino acid sequence of form e comprises a serine at position 425 and a valine at position 474; (G′) The amino acid sequence of form h is identical to SEQ ID NO: 2 except that the amino acid sequence of form h comprises a threonine at position 443 and a proline at position 619; (H′) The amino acid sequence of form aj is identical to SEQ ID NO: 2 except that the amino acid sequence of form aj comprises valine at position 474; and (I′) The amino acid sequence of form q is identical to SEQ ID NO: 2 except that the amino acid sequence of form q comprises a glycine at position 670.

The mature form of p is identical to the mature form of f and aj.

The mature form of c is identical to the mature form of q.

Further sequence analysis and 3D in silico modelling (see FIG. 1 ) revealed that selected variants also fulfilled the following selection criteria:—

-   -   PCSK9 variants whose variant amino acid residues (versus the         common form of human PCSK9) are found in the mature form of the         target (ie, outside the pro-domain); and     -   PCSK9 variants whose variant amino acid residues (versus the         common form of human PCSK9) are surface-exposed on the target,         which the inventor saw as contributing to determining the         topography of the target and potentially contributing to how and         where ligand binding on the target occurs.

As shown in FIG. 1 , identified positions 425, 443, 474, 619 and 670 (found in the selected variants of the invention) are all surface-exposed and outside of the pro-domain. Variant positions 425 and 443 are surface-exposed on the catalytic domain, while variant positions 474, 619 and 670 are surface-exposed on the C-terminal domain.

In a first example, the invention addresses the need to treat humans having naturally-occurring rarer natural PCSK9 alleles, genotypes and phenotypes (rarer protein forms). In this respect, the invention provides the following aspects:

In a First Aspect: An anti-human PCSK9 ligand for use in a method of treating and/or preventing a PCSK9-mediated disease or condition in a human whose genome comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, wherein the method comprises administering the ligand to the human.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37. These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In the alternative the nucleotide sequence encodes a human PCSK9 comprising a mutation R46L, A53V, N425S, A443T, I474V, Q619P and E670G (eg, comprises a mutation I474, E670G, N425S or Q619P) in SEQ ID NO: 1; for example, the PCSK9 comprises I474V in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises E670G in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises Q619P in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises N425S in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises R46L in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg increasing cholesterol in the human); for example, the PCSK9 comprises A53V in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg increasing cholesterol in the human); for example, the PCSK9 comprises A443T in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg increasing cholesterol in the human).

In a Second Aspect: The ligand of aspect 1, wherein the ligand has been or is determined as capable of binding a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q.

In the alternative the ligand has been or is determined as capable of binding a human PCSK9 comprising a mutation R46L, A53V, N425S, A443T, I474V, Q619P and E670G (eg, comprises a mutation I474, E670G, N425S or Q619P) in SEQ ID NO: 1; for example, the PCSK9 comprises I474V in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises E670G in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises Q619P in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises N425S in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg reducing cholesterol or maintaining reduced cholesterol in the human); for example, the PCSK9 comprises R46L in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg increasing cholesterol in the human); for example, the PCSK9 comprises A53V in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg increasing cholesterol in the human); for example, the PCSK9 comprises A443T in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein the method is for treating or preventing dislipidemia, eg increasing cholesterol in the human).

In an example of any aspect, the ligand binds (or has been determined to bind) two, three, four or more human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q or said PCSK9s in said alternative.

In an example of any aspect, the ligand comprises a protein domain that specifically binds to PCSK9, eg, a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q, or said PCSK9s in said alternative.

The term “specifically binds,” or the like, means that a ligand, eg, an antibody or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10⁻⁶ M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. An isolated antibody that specifically binds a human PCSK9 may, however, exhibit cross-reactivity to other antigens such as a PCSK9 molecule from another species. Moreover, multi-specific antibodies (e.g., bispecifics) that bind to human PCSK9 and one or more additional antigens are nonetheless considered antibodies that “specifically bind” PCSK9, as used herein.

In an example of any aspect, the ligand comprises or consists of a protein that mimics the EGFA domain of the LDL receptor and specifically binds to PCSK9, eg, a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q, or said PCSK9s in said alternative.

In an example of any aspect, the ligand antagonises PCSK9, eg, a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q, or said PCSK9s in said alternative.

In an example of any aspect, the method comprises (before administering the ligand) the step of determining that the ligand is capable of binding a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q, or said PCSK9s in said alternative.

In an example of any aspect, binding is determined by SPR. In an example of any aspect, binding is determined by ELISA.

In an example of any aspect, said forms are the mature forms.

In an example of any aspect, said forms are the pro-forms.

In a Third Aspect: A ligand that binds (i) a PCSK9 of said alternative or (ii) a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27 for use in a method comprising the step of using the ligand to target said PCSK9 in a human to treat and/or prevent a disease or condition mediated by PCSK9, the method comprising administering the ligand to the human.

In an example, the disease or condition is mediated by (i) a PCSK9 of said alternative or (ii) a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27.

In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the group consisting of SEQ ID NOs: 4-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 4-14, 18-23, 26 and 27. These are naturally-occurring sequences that do not comprise 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the amino acid sequence is SEQ ID NO: 18, 19 or 20, that comprises a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 10-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequences that have a naturally-occurring combination of differences from SEQ ID NOs: 1-3 (form a) and which meet the criteria set out above.

In an example, the amino acid sequence is SEQ ID NO: 4.

In an example, the amino acid sequence is SEQ ID NO: 5.

In an example, the amino acid sequence is SEQ ID NO: 6.

In an example, the amino acid sequence is SEQ ID NO: 7.

In an example, the amino acid sequence is SEQ ID NO: 8.

In an example, the amino acid sequence is SEQ ID NO: 9.

In an example, the amino acid sequence is SEQ ID NO: 10.

In an example, the amino acid sequence is SEQ ID NO: 11.

In an example, the amino acid sequence is SEQ ID NO: 12.

In an example, the amino acid sequence is SEQ ID NO: 13.

In an example, the amino acid sequence is SEQ ID NO: 14.

In an example, the amino acid sequence is SEQ ID NO: 15.

In an example, the amino acid sequence is SEQ ID NO: 16.

In an example, the amino acid sequence is SEQ ID NO: 17.

In an example, the amino acid sequence is SEQ ID NO: 18.

In an example, the amino acid sequence is SEQ ID NO: 19.

In an example, the amino acid sequence is SEQ ID NO: 20.

In an example, the amino acid sequence is SEQ ID NO: 21.

In an example, the amino acid sequence is SEQ ID NO: 22.

In an example, the amino acid sequence is SEQ ID NO: 23.

In an example, the amino acid sequence is SEQ ID NO: 24.

In an example, the amino acid sequence is SEQ ID NO: 25.

In an example, the amino acid sequence is SEQ ID NO: 26.

In an example, the amino acid sequence is SEQ ID NO: 27.

In a Fourth Aspect: The ligand of aspect 3, wherein the genome of the human comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37; or a nucleotide sequence encoding a PCSK9 of said alternative.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37. These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33

In an example, the nucleotide sequence is SEQ ID NO: 34

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Fifth Aspect: The ligand of any preceding aspect, wherein the human has been or is genotyped as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37. These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Sixth Aspect: The ligand of any preceding aspect, wherein the human has been or is phenotyped as positive for (i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q or at least the catalytic or C-terminal domain thereof.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Seventh Aspect: The ligand of any preceding aspect, wherein the method comprises genotyping the human as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof; or a nucleotide sequence encoding a PCSK9 of said alternative.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37. These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an Eighth Aspect: The ligand of any preceding aspect, wherein the method comprises phenotyping the human has positive for (i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q or at least the catalytic or C-terminal domain thereof.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Ninth Aspect: The ligand of any preceding aspect, wherein the human has been or is genotyped as heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof; or a nucleotide sequence encoding a PCSK9 of said alternative; optionally wherein the human has been or is genotyped as comprising the nucleotide sequence of SEQ ID NO: 28 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof; or a nucleotide sequence encoding a PCSK9 of said alternative.

“Heterozygous” here means that in the human's genotype one allele comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof and other allele can be any PCSK9 (eg, form a, a′ or an allele comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof).

In an example, the method comprises (before administering the ligand) genotyping the human as heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof; optionally also genotyping the human as comprising the nucleotide sequence of SEQ ID NO: 28 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37.

These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Tenth Aspect: The ligand of any one of aspects 1 to 9, wherein the genome of the human has been or is genotyped as homozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof; or a nucleotide sequence encoding a PCSK9 of said alternative.

“Homozygous” here means that in the human's genotype each allele comprises the same nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.

In an example, the method comprises genotyping the human as homozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof; or a nucleotide sequence encoding a PCSK9 of said alternative.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37. These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an Eleventh Aspect: The ligand of any preceding aspect, wherein the ligand comprises an antibody binding site that binds (i) a PCSK9 of said alternative or (ii) a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27 and optionally has been or is determined as capable of such binding.

In an example, the method comprises (before administering the ligand) the step of determining that the ligand is capable of binding to said human PCSK9.

In an example, the binding is specific binding. In an example, the ligand binds (or has been determined as binding) to the PCSK9 with an affinity (Kd) of 1 mM, 100 nM, 10 nM or 1 nM or less. In an embodiment, the affinity is no less than 10, 100 or 1000 fM.

In an example, binding or affinity is determined by SPR or ELISA.

In an example, the disease or condition is mediated by a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27.

In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the group consisting of SEQ ID NOs: 4-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 4-14, 18-23, 26 and 27. These are naturally-occurring sequences that do not comprise 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the amino acid sequence is SEQ ID NO: 18, 19 or 20, that comprises a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 10-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequences that have a naturally-occurring combination of differences from SEQ ID NOs: 1-3 (form a) and which meet the criteria set out above

In an example, the amino acid sequence is SEQ ID NO: 4.

In an example, the amino acid sequence is SEQ ID NO: 5.

In an example, the amino acid sequence is SEQ ID NO: 6.

In an example, the amino acid sequence is SEQ ID NO: 7.

In an example, the amino acid sequence is SEQ ID NO: 8.

In an example, the amino acid sequence is SEQ ID NO: 9.

In an example, the amino acid sequence is SEQ ID NO: 10.

In an example, the amino acid sequence is SEQ ID NO: 11.

In an example, the amino acid sequence is SEQ ID NO: 12.

In an example, the amino acid sequence is SEQ ID NO: 13.

In an example, the amino acid sequence is SEQ ID NO: 14.

In an example, the amino acid sequence is SEQ ID NO: 15.

In an example, the amino acid sequence is SEQ ID NO: 16.

In an example, the amino acid sequence is SEQ ID NO: 17.

In an example, the amino acid sequence is SEQ ID NO: 18.

In an example, the amino acid sequence is SEQ ID NO: 19.

In an example, the amino acid sequence is SEQ ID NO: 20.

In an example, the amino acid sequence is SEQ ID NO: 21.

In an example, the amino acid sequence is SEQ ID NO: 22.

In an example, the amino acid sequence is SEQ ID NO: 23.

In an example, the amino acid sequence is SEQ ID NO: 24.

In an example, the amino acid sequence is SEQ ID NO: 25.

In an example, the amino acid sequence is SEQ ID NO: 26.

In an example, the amino acid sequence is SEQ ID NO: 27.

In a Twelfth Aspect: The ligand of aspect 11, wherein the ligand is an antibody or antibody fragment. For example, the antibody or antibody fragment is a PCSK9 antagonist, eg, neutralises PCSK9.

Examples of such antibodies are disclosed, for instance, in WO 2008/057457, WO2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/133647, WO 2009/100297, WO 2009/100318, WO 201 1/037791, WO 201 1/053759, WO 201 1/053783, WO 2008/125623, WO 2011/072263, WO 2009/055783, WO 2010/029513, WO 2011/11 1007, WO 2010/077854, the disclosures and sequences of such antibodies being incorporated herein for use in the invention in their entireties by reference. One specific example is AMG 145 (Amgen), LY3015014 (Eli Lilly) or alirocumab. Advantageously, the ligand is or comprises alirocumab. Alternatively, the ligand is or comprises evolocumab.

In an example, the ligand is SAR236553/REGN727 (Sanofi Aventis/Regeneron) or a PCSK9-binding derivative thereof.

In an example, the ligand comprises or consists of a neutralizing antibody that binds to the PCSK9, wherein the antibody binds to PCSK9 and reduces the likelihood that PCSK9 binds to LDLR.

The ligand of aspect 11, wherein the ligand is a PCSK9 antagonist, eg, neutralises PCSK9.

In an example of any aspect of the invention, the ligand comprises or consists a ligand selected from evolocumab, 1D05-IgG2 (Merck & Co.), ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat), LY3015014 (Eli Lilly) and alirocumab (SAR236553/REGN727; Sanofi Aventis/Regeneron).

In Thirteenth Aspect: The ligand of any one of aspects 1 to 10, wherein (i) the ligand comprises a sequence of contiguous nucleotides that specifically hybridises to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof (or hybridizes to a nucleotide sequence encoding a PCSK9 of said alternative), or specifically hybridises to an antisense sequence or an RNA transcript of said sequence, wherein said sequence of contiguous nucleotides hybridises to at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28 or hybridises to an antisense sequence or an RNA transcript thereof respectively; and/or (ii) the ligand comprises a sequence of at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 thereof (or a nucleotide sequence encoding a PCSK9 of said alternative) or is an antisense sequence or RNA version of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37. These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an embodiment, the ligand comprises at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or 100 contiguous nucleotides of said nucleotide sequence.

In a Fourteenth Aspect: The ligand of any preceding aspect, wherein said disease or condition is hyperlipidaemia, hypercholesterolaemia (eg, familial hypercholesterolaemia), heart attack, stroke, coronary heart disease, atherosclerosis or a cardiovascular disease or condition.

The ligand of any preceding aspect, wherein the disease or condition is hypercholesterolemia, hyperlipidemia, hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

In an example, said disease or condition is hypercholesterolaemia. The term “hypercholesterolaemia,” as used herein, refers to a condition in which cholesterol levels are elevated above a desired level. In some embodiments, this denotes that serum cholesterol levels are elevated. In some embodiments, the desired level takes into account various “risk factors” that are known to one of skill in the art (and are described or referenced in US20120093818).

The ligand of any preceding aspect, wherein the human is identified as heterozygous for Familial Hypercholesterolemia, statin intolerant, statin uncontrolled, or at risk for developing hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

In a Fifteenth Aspect: The ligand of any preceding aspect, wherein said disease or condition is associated with elevated LDL cholesterol.

Cholesterol levels are measured in milligrams (mg) of cholesterol per deciliter (dL) of blood in the United States and some other countries. Canada and most European countries measure cholesterol in millimoles (mmol) per liter (L) of blood. Below are general guideline ideal ranges and elevated ranges.

Total cholesterol (U.S. and some Total cholesterol* other countries) (Canada and most of Europe) Below 200 mg/dL Below 5.2 mmol/L Ideal 200-239 mg/dL 5.2-6.2 mmol/L Borderline high 240 mg/dL and above Above 6.2 mmol/L High LDL cholesterol (U.S. and some LDL cholesterol* other countries) (Canada and most of Europe) 100-129 mg/dL 2.6-3.3 mmol/L Ideal 130-159 mg/dL 3.4-4.1 mmol/L Borderline high 160-189 mg/dL 4.1-4.9 mmol/L High 190 mg/dL and above Above 4.9 mmol/L Very high *Canadian and European guidelines differ slightly from U.S. guidelines. These conversions are based on U.S. guidelines.

Elevated LDL cholesterol is, therefore, 160 mg/dL or above (4.1 mmol/L or above).

In a Sixteenth Aspect: The ligand of any preceding aspect, wherein the ligand inhibits human PCSK9 binding to human LDL receptor and optionally has been or is determined as capable of such inhibition.

In an example, the method comprises (before administering the ligand) determining that the ligand is capable of such inhibition.

Inhibition determination is eg, inhibition in a blood or serum sample, at rtp, at ph7, at 37 degrees centigrade and/or under the physiological conditions of a human body.

In a Seventeeth Aspect: The ligand of any preceding aspect, wherein the human is resistant or substantially resistant to statin (eg, avorstatin and/or fluvastatin) treatment of said disease or condition.

In an Eighteenth Aspect: The ligand of any preceding aspect, wherein the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human (i) whose genome comprises SEQ ID NO: 29 and wherein the human is of ASW, YRI, GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry; or

(ii) whose genome comprises SEQ ID NO: 30 and wherein the human is of ASW, YRI, GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry; or (iii) whose genome comprises SEQ ID NO: 32 and wherein the human is of ASW, GBR, TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry; or (iv) whose genome comprises SEQ ID NO: 33 and wherein the human is of LWK, ASW, YRI or CLM ancestry; or (v) whose genome comprises SEQ ID NO: 34 and wherein the human is of LWK, ASW or YRI ancestry; or (vi) whose genome comprises SEQ ID NO: 35 and wherein the human is of PUR, TSI, FIN or CEU ancestry; or (vii) whose genome comprises SEQ ID NO: 36 and wherein the human is of LWK, ASW or YRI ancestry; or (viii) whose genome comprises SEQ ID NO: 37 and wherein the human is of CHS, ASW, JPT, PUR or CHB ancestry.

In a Nineteenth Aspect: The ligand of any preceding aspect, wherein the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human

(i) that expresses PCSK9 form f and wherein the human is of ASW, YRI, GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry; or (ii) that expresses PCSK9 form c and wherein the human is of ASW, YRI, GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry; or (iii) that expresses PCSK9 form p and wherein the human is of ASW, GBR, TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry; or (iv) that expresses PCSK9 form m and wherein the human is of LWK, ASW, YRI or CLM ancestry; or (v) that expresses PCSK9 form e and wherein the human is of LWK, ASW or YRI ancestry; or (vi) that expresses PCSK9 form h and wherein the human is of PUR, TSI, FIN or CEU ancestry; or (vii) that expresses PCSK9 form aj and wherein the human is of LWK, ASW or YRI ancestry; or (viii) that expresses PCSK9 form q and wherein the human is of CHS, ASW, JPT, PUR or CHB ancestry.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twentieth Aspect: A pharmaceutical composition or kit for treating and/or preventing a PCSK9-mediated condition or disease (eg, as recited in aspect 14 or 15), the composition or kit comprising a ligand of any preceding aspect and optionally a statin (eg, cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin or pravastatin); and optionally in combination with a label or instructions for use to treat and/or prevent said disease or condition in a human (eg, covering treatment of a human as recited in aspect 18 or 19); optionally wherein the label or instructions comprise a marketing authorisation number (eg, an FDA or EMA authorisation number); optionally wherein the label or instructions comprise directions to administer alirocumab or evolocumab to said human; optionally wherein the kit comprises an IV or injection device that comprises the ligand (and, eg, also a statin).

In a Twenty-first Aspect: A method of producing an anti-human PCSK9 antibody binding site, the method comprising obtaining a plurality of anti-PCSK9 antibody binding sites, screening the antibody binding sites for binding to (i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibody binding site that binds in the screening step, and optionally producing a form f, c, r, p, m, e, h, aj or q PCSK9-binding fragment or derivative of the isolated antibody.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In an example of this and the next aspect, the plurality of binding sites comprises or consists of a plurality of 4-chain antibodies or fragments thereof, eg, dAbs, Fabs or scFvs. Suitable methods for producing pluralities of binding sites for screening include phage display (producing a phage display library of antibody binding sites), ribosome display (producing a ribosome display library of antibody binding sites), yeast display (producing a yeast display library of antibody binding sites), or immunisation of a non-human vertebrate (eg, a rodent, eg, a mouse or rat, eg, a Velocimouse™, KymOuSe™, Xenomouse™, Aliva Mouse™, HuMab Mouse™, Omnimouse™ Omnirat™ or MeMo Mouse™) with a PCSK9 epitope and isolation of a repertoire of antibody-producing cells (eg, a B-cell, plasma cell or plasmablast repertoire) and/or a repertoire of isolated antibodies.

In an example, the method comprises selecting one or more antibody binding sites that each specifically binds to a human PCSK9 epitope comprising amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3.

In a Twenty-second Aspect: A method of producing an anti-human PCSK9 antibody, the method comprising immunising a non-human vertebrate (eg, a mouse or a rat) with (i) a PCSK9 of said alternative or (ii) a human PCSK9 comprising an amino acid sequence selected from the group consisting of the amino acid sequences of forms f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibody that binds a human PCSK9 comprising selected from the group consisting of forms f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3, and optionally producing a form f, c, r, p, m, e, h, aj or q PCSK9-binding fragment or derivative of the isolated antibody.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twenty-third Aspect: The method of aspect 21 or 22, comprising the step of obtaining a nucleic acid encoding the antibody, fragment, derivative or binding site and optionally inserting the nucleic acid in an expression vector.

For example, the method comprises isolating a cell (eg, B-cell, plasmablast, plasma cell or memory cell) comprising the nucleic acid, wherein the cell is obtained from a non-human vertebrate that has been immunised with the PCSK9 epitope.

In a Twenty-fourth Aspect: A kit for PCSK9 genotyping a human, wherein the kit comprises a nucleic acid (i) comprising a sequence of 10 or more (eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) contiguous nucleotides that specifically hybridises to (i) a nucleotide sequence encoding a PCSK9 of said alternative or (ii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof, or specifically hybridises to an antisense sequence or an RNA transcript of said sequence, wherein said sequence of contiguous nucleotides hybridises to at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28 or hybridises to an antisense sequence or an RNA transcript thereof; and/or (ii) comprising a sequence of at least 10 or more (eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 (or the nucleotide sequence of (i)) or comprising an antisense sequence or RNA version of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37. These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Twenty-fifth Aspect: A kit for PCSK9 genotyping or phenotyping a human, wherein the kit comprises a ligand according to any one of aspects 1 to 19 or an antibody, fragment or derivative produced by the method of any one of aspects 21 to 23.

In a Twenty-sixth Aspect: Use of an anti-PCSK9 ligand that binds (i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q in the manufacture of a medicament for treating and/or preventing a PCSK9-mediated disease or condition in a human whose genome comprises (iii) a nucleotide sequence encoding said PCSK9 of (i); or (iv) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, optionally for treating and/or preventing a PCSK9-mediated disease or condition in a human as recited in aspect 18 or 19.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twenty-seventh Aspect: Use of an anti-PCSK9 ligand that binds (i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q in the manufacture of a medicament for targeting said PCSK9 in a human to treat and/or prevent a disease or condition mediated by PCSK9, optionally for targeting PCSK9 in a human as recited in aspect 18 or 19.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37. These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Twenty-eight Aspect: The use of aspect 26 or 27, wherein the ligand, human, disease or condition is according to any one of aspects 1 to 19.

In a Twenty-ninth Aspect: A method of targeting a PCSK9 for treating and/or preventing a PCSK9-mediated disease or condition in a human, the method comprising administering an anti-PCSK9 ligand to a human comprising (i) a nucleotide sequence encoding a PCSK9 of said alternative or (ii) a nucleotide sequence selected from the group consisting SEQ ID NOs: 29-37, whereby a PCSK9 encoded by said nucleotide sequence is targeted.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Thirtieth Aspect: The method of aspect 29, wherein the method comprises targeting a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q with said ligand to treat and/or prevent said disease or condition in said human.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-first Aspect: A method of treating and/or preventing a disease or condition mediated by PCSK9 in a human, the method comprising targeting (i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q by administering to the human a ligand that binds said PCSK9 thereby treating and/or preventing said disease or condition in the human.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Thirty-second Aspect: The method of aspect 31, wherein the genome of the human comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37.

In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37. These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Thirty-third Aspect: The method of any one of aspects 29 to 32, wherein the human has been or is genotyped as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.

In a Thirty-fourth Aspect: The method of any one of aspects 29 to 33, wherein the human has been or is phenotyped as positive for (i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-fifth Aspect: The method of any one of aspects 29 to 34, wherein the method comprises genotyping the human as positive for (i) a nucleotide sequence encoding a PCSK9 of said alternative or (ii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.

In a Thirty-sixth Aspect: The method of any one of aspects 29 to 35, wherein the method comprises phenotyping the human as positive for (i) a PCSK9 of said alternative or (ii) a human PCSK9 sequence selected from the group consisting of forms f, c, r, p, m, e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-seventh Aspect: The method of any one of aspects 29 to 36, wherein the human has been or is genotyped as heterozygous for said nucleotide sequence of (i) or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof; optionally wherein the human has been or is genotyped as comprising the nucleotide sequence of SEQ ID NO: 28 or the catalytic- or C-terminal domain-encoding sequence thereof and said nucleotide sequence of (i) or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.

In a Thirty-eighth Aspect: The method of any one of aspects 29 to 37, wherein the genome of the human has been or is genotyped as homozygous for said nucleotide sequence of (i) or a nucleotide sequence selected from the group consisting of of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.

In a Thirty-ninth Aspect: The method of any one of aspects 29 to 38, wherein the method comprises genotyping the human for said nucleotide sequence of (i) or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof before administering the ligand to the human, wherein the ligand is determined to be capable of binding to a PCSK9 encoded by said selected sequence.

In a Fortieth Aspect: The method of any one of aspects 29 to 39, wherein the ligand, human, disease or condition is according to any one of aspects 1 to 19.

In a Forty-first Aspect: A method according to any one of aspects 29 to 40 for treating and/or preventing a condition or disease as recited in aspect 14 or 15, the method comprising administering said ligand and a statin (eg, cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin or pravastatin) to the human.

In a Forty-second Aspect: The method of aspect 41, wherein the ligand and statin are administered separately.

In a Forty-third Aspect: The method of aspect 41, wherein the ligand and statin are administered simultaneously.

In a Forty-fourth Aspect: The method of any one of aspects 29 to 43, wherein the ligand is administered by subcutaneous injection.

In a Forty-fifth Aspect: A method of PCSK9 genotyping a nucleic acid sample of a human, the method comprising identifying in the sample the presence of (i) a nucleotide sequence encoding a PCSK9 of said alternative or (ii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.

In a Forty-sixth Aspect: A method of PCSK9 typing a protein sample of a human, the method comprising identifying in the sample the presence of (i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Forty-seventh Aspect: The method of aspect 45 or 46, comprising obtaining a sample of serum, blood, faeces, hair, tissue, cells, urine or saliva from a human, whereby the nucleic acid or protein sample is obtained and used in the step of identifying said sequence.

In a Forty-eighth Aspect: The method of any one of aspects 45 to 47, comprising using a ligand according to any one of aspects 1 to 19 to carry out said identifying step.

In a Forty-ninth Aspect: A method of treating and/or preventing in a human patient a cardiovascular disease or condition, or a disease or condition that is associated with elevated LDL cholesterol (eg, hypercholesterolaemia), wherein the patient is receiving or has previously received statin treatment for said disease or condition, the method comprising typing the patient using a method of any one of aspects 45 to 48 and administering a ligand according to one of aspects 1 to 19 whereby the human is treated or said disease or condition is prevented; optionally also reducing or stopping statin treatment.

In an example, said reducing or stopping comprises reducing the dose and/or dosing frequency of statin.

In a Fiftieth Aspect: A diagnostic, therapeutic or prophylactic kit comprising a ligand that is capable of binding to or has been or is determined as capable of binding to an amino acid sequence selected from SEQ ID NOs: 4-27 and instructions for carrying out the method of any one of aspects 46 to 49 and/or a label or instructions indicating or covering administration of the ligand to a human as defined in any one of aspects 1 to 19.

In a Fifty-first Aspect: A diagnostic, therapeutic or prophylactic kit comprising a nucleic acid probe comprising a nucleotide sequence that specifically hybridises to a nucleotide sequence or (i) or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or an antisense sequence or RNA transcript thereof and instructions for carrying out the method of aspect 45, 47 or 48.

In examples of the present invention, the ligand specifically binds to human PCSK9, eg, one or more of (i) the PCSK9s of said alternative or (ii) the rare PCSK9 variants disclosed herein (eg, one, two, three, more or all mature forms f, c, r, p, m, e, h, aj and q) and optionally also the a and/or a′ form. For example, the ligand specifically binds to mature form f and/or c as well as form a. For example, the ligand specifically binds to a human PCSK9 comprising a mutation E670G and also binds to a human PCSK9 comprising a mutation I474V, Q619P or N425S in SEQ ID NO: 1. For example, the ligand specifically binds to a human PCSK9 comprising a mutation E670G and also binds to a human PCSK9 comprising a mutation I474V in SEQ ID NO: 1. For example, the ligand specifically binds to a human PCSK9 comprising a mutation E670G and also binds to a human PCSK9 comprising a mutation Q619P in SEQ ID NO: 1. For example, the ligand specifically binds to a human PCSK9 comprising a mutation E670G and also binds to a human PCSK9 comprising a mutation N425S in SEQ ID NO: 1. Determination of such binding can be performed by any antibody binding test as known in the art, eg, by surface plasmon resonance. Binding to each such form is, for example, respectively with a Kd of at least 1 mM, 100 nM, 1 nM, 100 pM, 10 pM or 1 pM.

In an example, the ligand binds form a and (i) a PCSK9 of said alternative or (ii) a PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q, wherein the ligand binding to said selected form is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form f wherein the ligand binding to form f is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form c, wherein the ligand binding to form c is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form r, wherein the ligand binding to form r is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form p, wherein the ligand binding to form p is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form m, wherein the ligand binding to form m is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form e, wherein the ligand binding to form e is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form h, wherein the ligand binding to form h is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form aj, wherein the ligand binding to form aj is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form q, wherein the ligand binding to form q is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

In examples of the present invention, the ligand neutralises human PCSK9, eg, one or more of the rare PCSK9 variants disclosed herein (eg, one, two, three, more or all mature forms f, c, r, p, m, e, h, aj and q) and optionally also the a and/or a′ form. For example, the ligand neutralises mature form f and/or c as well as form a. Determination of neutralisation can be performed, for example, by any neutralisation assay method disclosed in US20120093818A1 (Amgen, Inc) or US20110065902A1 (Regeneron Pharmaceuticals, Inc). Ligands of the invention that bind or target PCSK9 are useful, for example, for therapeutic and prophylactic applications disclosed in US20120093818A1 and US20110065902A1, these specific disclosures being incorporated herein by reference for use in the present invention and for possible inclusion in claims herein.

In embodiments where the ligand is used for therapeutic applications, an antigen binding protein can inhibit, interfere with or modulate one or more biological activities of a PCSK9 (eg, one or more of the rare variants disclosed herein and optionally also the a and/or a′ form). In one embodiment, ligand binds specifically to human PCSK9 (eg, one or more of the rare variants disclosed herein and optionally also the a and/or a′ form) and/or substantially inhibits binding of human PCSK9 (eg, said one or more of the rare variants disclosed herein and optionally also the a and/or a′ form) to LDLR by at least 20%, eg, 20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuring binding in an in vitro competitive binding assay). In an example, the ligand is an antibody.

In an embodiment, the ligand has a Kd of less (binding more tightly) than 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ M for binding to one, two or more of the rare variants disclosed herein and optionally also the a and/or a′ form. In an example, Kd is determined using SPR.

In an embodiment, the ligand has an IC50 for blocking the binding of LDLR to one or more of the rare PCSK9 variants disclosed herein (and optionally also the a and/or a′ form) of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM.

In an embodiment, the ligand has an IC50 for blocking the binding of LDLR to the a and/or a′ form of PCSK9 that is no more than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10-fold more (ie, more inhibitory) than the IC50 for blocking the binding of LDLR to one or more of the rare PCSK9 variants disclosed herein (eg, one or more PCSK9 proteins comprising a sequence selected from SEQ ID NOs: 4 to 27). Additionally or alternatively, for example, the ligand has an IC50 for blocking the binding of LDLR to (i) the a and/or a′ form of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, eg, in the range of 1 mM to 1 pM (eg, 1 mM to 100 pM; 10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1 pM) and (ii) one or more PCSK9 proteins comprising a sequence selected from SEQ ID NOs: 4 to 27 of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, eg, in the range of 1 mM to 1 pM (eg, 1 mM to 100 pM; 10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1 pM).

In an embodiment, the ligand binds to the a and/or a′ form of PCSK9 with a binding affinity (Kd) that is greater than up to 10%, greater than up to 20%, greater than up to 40%, greater than up to 50%, greater than up to 55%, greater than up to 60%, greater than up to 65%, greater than up to 70%, greater than up to 75%, greater than up to 80%, greater than up to 85%, greater than up to 90%, greater than up to 95% or greater than up to 100% (ie, is double) relative to binding to a PCSK9 comprising a sequence selected from SEQ ID NOs: 4 to 27. Such binding measurements can be made using a variety of binding assays known in the art, eg, using surface plasmon resonance (SPR), such as by Biacore™ or using the ProteOn XPR36™ (Bio-Rad®), or using KinExA® (Sapidyne Instruments, Inc).

In one embodiment, the surface plasmon resonance (SPR) is carried out at 25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such as about pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (also referred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level, eg, 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of no greater than 0.05% by volume, eg, in the presence of P20 (polysorbate 20; eg, Tween-20™) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a buffer at pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. The buffer can contain 10 mM Hepes. In one example, the SPR is carried out at 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc (California; catalogue number H8022).

In an example, the affinity of the ligand which is an antibody is determined using SPR by

1. Coupling anti-mouse (or other relevant vertebrate) IgG (eg, Biacore BR-1008-38) to a biosensor chip (eg, GLM chip) such as by primary amine coupling; 2. Exposing the anti-mouse IgG (vertebrate antibody) to a test IgG antibody to capture test antibody on the chip; 3. Passing the test antigen over the chip's capture surface at 1024 nM, 256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and 4. And determining the affinity of binding of test antibody to test antigen using surface plasmon resonance, eg, under an SPR condition discussed above (eg, at 25° C. in physiological buffer). SPR can be carried out using any standard SPR apparatus, such as by Biacore™ or using the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mM glycine at pH1.7. This removes the captured antibody and allows the surface to be used for another interaction. The binding data can be fitted to 1:1 model inherent using standard techniques, eg, using a model inherent to the ProteOn XPR36™ analysis software.

In an embodiment, assaying or testing of a ligand of the invention is carried out at or substantially at pH7 (eg, for in vitro tests and assays) and at or substantially at rtp.

One example of an IgG2 heavy chain constant domain of an anti-PCSK9 antibody of the present invention has the amino acid sequence as shown in SEQ ID NO: 154, FIG. 3KK of US20120093818A1, which sequence is incorporated herein by reference.

One example of an IgG4 heavy chain constant domain of an anti-PCSK9 antibody of the present invention has the amino acid sequence as shown in SEQ ID NO: 155, FIG. 3KK of US20120093818A1, which sequence is incorporated herein by reference.

One example of a kappa light chain constant domain of an anti-PCSK9 antibody has the amino acid sequence as shown in SEQ ID NO: 157, FIG. 3KK which sequence is incorporated herein by reference.

One example of a lambda light chain constant domain of an anti-PCSK9 antibody has the amino acid sequence as shown in SEQ ID NO: 156, FIG. 3KK of US20120093818A1, which sequence is incorporated herein by reference.

In examples of the present invention, the ligand binds mature PCSK9, eg, a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a′ form.

In examples of the present invention, the ligand binds the catalytic domain of PCSK9, eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a′ form.

In examples of the present invention, the ligand binds the prodomain of PCSK9, eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a′ form.

In some embodiments, the ligand binds to the V domain of PCSK9, eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a′ form. In some embodiments, the ligand binds to the V domain of PCSK9 (eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a′ form) and prevents (or reduces, eg, by at least 10%) PCSK9 from binding to LDLR. In some embodiments, the ligand binds to the V domain of PCSK9 (eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a′ form), and while it does not prevent (or reduce) the binding of PCSK9 to LDLR, the ligand prevents or reduces (eg, by at least 10%) the adverse activities mediated through PCSK9 on LDLR.

In examples of the present invention, the ligand is or comprises a fully human antibody. In an example, the ligand comprises human variable regions or humanised variable regions.

In an example, the ligand of the invention specifically binds to an epitope of (i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q, wherein the epitope comprises at least one amino acid that is not found in form a. For example, the amino acid is selected from the group consisting of 46L, 53V, 425S, 443T, 474V, 619P and 670G (numbering as used in SEQ ID NO:1). For example, the amino acid is selected from the group consisting of 425S, 443T, 474V, 619P and 670G (numbering as used in SEQ ID NO:1). For example, the amino acid is selected from the group consisting of 425S and 443T (numbering as used in SEQ ID NO:1). For example, the amino acid is selected from the group consisting of 474V, 619P and 670G (numbering as used in SEQ ID NO:1). In an example, the PCSK9 form is the mature form. In an example, the PCSK9 form is the pro-form. In an example, the ligand also specifically binds to form a and/or a′. In an embodiment, the ligand specifically binds to an epitope of form f PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form aj PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the pro-domain of a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q. In an example, the ligand also specifically binds to the pro-domain of form a and/or a′. In an embodiment, the ligand specifically binds to the pro-domain of form f PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form aj PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the catalytic domain of a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q. In an example, the ligand also specifically binds to the catalytic domain of form a and/or a′. In an embodiment, the ligand specifically binds to the catalytic domain of form f PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form aj PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the C-terminal domain of a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q. In an example, the ligand also specifically binds to the C-terminal domain of form a and/or a′. In an embodiment, the ligand specifically binds to the C-terminal domain of form f PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form aj PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the substrate-binding groove of a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q (see Cunningham et al., Nat Struct Mol Biol. 2007 May; 14(5):413-9. Epub 2007 Apr. 15, “Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia”, incorporated herein in its entirety by reference). In an example, the ligand also specifically binds to the substrate-binding groove of form a and/or a′. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form f PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form aj PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.

Reference is made to US20120093818A1 (Amgen, Inc), the entire disclosure of which is incorporated herein. This patent application discloses relevant ligands for use in the present invention, as well as examples and methods of producing and testing ligands that can be used with reference to the present invention.

In an example, the ligand is or comprises an antibody disclosed in Table 2 of US20120093818A1 (Amgen, Inc) or is a PCSK9-binding derivative thereof.

In an embodiment, the PCSK9-binding ligand of the invention is selected from the antigen binding proteins disclosed in US20120093818A1 (Amgen, Inc), eg, in paragraphs [0009] to [0014] and [0058] to [0063] of US20120093818A1; all of these disclosures (including the sequences of such proteins) are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention.

In this paragraph SEQ ID NOs are those as appearing in US20120093818A1 (Amgen, Inc) and these sequences are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention. In some aspects, the ligand of the invention comprises an isolated antigen binding protein that binds PCSK9 comprising: A) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 from a CDRH1 in a sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii) a CDRH2 from a CDRH2 in a sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (iii) a CDRH3 from a CDRH3 in a sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; and (iv) a CDRH of (i), (ii), and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 4 amino acids; B) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 from a CDRL1 in a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (ii) a CDRL2 from a CDRL2 in a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (iii) a CDRL3 from a CDRL3 in a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; and (iv) a CDRL of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 4 amino acids; or C) one or more heavy chain CDRHs of A) and one or more light chain CDRLs of B). In some embodiments, the isolated antigen binding protein comprises at least one CDRH of A) and at least one CDRL of B). In some embodiments, the isolated antigen binding protein comprises at least two CDRH of A) and at least two CDRL of B). In some embodiments, the isolated antigen binding protein comprises said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3. In some embodiments, the CDRH of A) is selected from at least one of the group consisting of: (i) a CDRH1 amino acid sequence selected from the CDRH1 in a sequence selected from the group consisting of SEQ ID NO: 67, 79, 89, and 49; (ii) a CDRH2 amino acid sequence selected from the CDRH2 in a sequence selected from the group consisting of SEQ ID NO: 67, 79, 89, and 49; (iii) a CDRH3 amino acid sequence selected from the CDRH3 in a sequence selected from the group consisting of SEQ ID NO: 67, 79, 89, and 49; and (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids. In addition, the CDRL of B) is selected from at least one of the group consisting of: (i) a CDRL1 amino acid sequence selected from the CDRL1 in a sequence selected from the group consisting of SEQ ID NO: 12, 35, 32, and 23; (ii) a CDRL2 amino acid sequence selected from the CDRL2 in a sequence selected from the group consisting of SEQ ID NO: 12, 35, 32, and 23; (iii) a CDRL3 amino acid sequence selected from the CDRL3 in a sequence selected from the group consisting of SEQ ID NO: 12, 35, 32, and 23; and (iv) a CDRL of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; or C) one or more heavy chain CDRHs of A) and one or more light chain CDRLs of B. In some embodiments, the CDRH of A) is selected from at least one of the group consisting of: (i) a CDRH1 amino acid sequence of the CDRH1 amino acid sequence in SEQ ID NO: 67; (ii) a CDRH2 amino acid sequence of the CDRH2 amino acid sequence in SEQ ID NO: 67; (iii) a CDRH3 amino acid sequence of the CDRH3 amino acid sequence in SEQ ID NO: 67; and (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; said CDRL of B) is selected from at least one of the group consisting of: (i) a CDRL1 amino acid sequence of the CDRL1 amino acid sequence in SEQ ID NO: 12; (ii) a CDRL2 amino acid sequence of the CDRL2 amino acid sequence in SEQ ID NO: 12; (iii) a CDRL3 amino acid sequence of the CDRL3 amino acid sequence in SEQ ID NO: 12; and (iv) a CDRL of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; or C) one or more heavy chain CDRHs of A) and one or more light chain CDRLs of B). In some embodiments, the antigen binding protein comprises A) a CDRH1 of the CDRH1 sequence in SEQ ID NO: 67, a CDRH2 of the CDRH2 sequence in SEQ ID NO: 67, and a CDRH3 of the CDRH3 sequence in SEQ ID NO: 67, and B) a CDRL1 of the CDRL1 sequence in SEQ ID NO: 12, a CDRL2 of the CDRL2 sequence in SEQ ID NO: 12, and a CDRL3 of the CDRL3 sequence in SEQ ID NO: 12. In some embodiments, the antigen binding protein comprises a heavy chain variable region (VH) having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or a light chain variable region (VL) having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, the VH has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or the VL has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, the VH is selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or the VL is selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46.

In an example of any aspect of the invention, the PCSK9-targeting or binding ligand comprises or consists of AMG145 or 31H4, 16F12, 11F1, 8A3 or 21B12 disclosed in US20120093818A1 (Amgen, Inc) or an antibody comprising the variable domains of AMG145, 31H4, 16F12, 11F1, 8A3 or 21B12, the disclosures of which (including sequences) are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention. Preferably, the PCSK9-targeting or binding ligand comprises or consists of AMG145.

In an example, the AMG145 or other ligand of the invention is glycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos or Hek293 cell). In an example, the ligand of the invention is produced in CHO.

Reference is made to US20110065902A1 (Regeneron Pharmaceuticals, Inc), the entire disclosure of which is incorporated herein. This patent application discloses relevant ligands for use in the present invention, as well as examples and methods of producing and testing ligands and determining medical efficacy that can be used with reference to the present invention.

Reference is made to the following PCT applications, the entire disclosures of which are incorporated herein. These disclose relevant ligands for use in the present invention, as well as examples and methods of producing and testing ligands and determining medical efficacy that can be used with reference to the present invention.

WO2008057457 WO2008057458 WO2008057459 WO2008063382 WO2008133647 WO2009100297 WO2009100318 WO2011037791 WO2011053759 WO2011053783 WO2008125623 WO2011072263 WO2009055783 WO2010029513 WO2011111007 WO2010077854

Antibody ligands to PCSK9 are described in, for example, WO 2008/057457, WO 2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/125623, and US 2008/0008697; each of which is incorporated by reference herein in its entirety.

In an example, the ligand is or comprises an antibody disclosed in the Examples of US20110065902A1 (eg, 316P or 300N) or is a PCSK9-binding derivative thereof. All of these disclosures (including the sequences of such proteins and corresponding nucleotide sequences) are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention. In an embodiment, the ligand is or comprises the variable domains of antibody 316P or 300N disclosed in US20110065902A1 or is (or comprises) such antibody or a PCSK9-binding derivative thereof.

In an embodiment, the ligand is or comprises the variable domains of antibody alirocumab or SAR236553/REGN727 (Sanofi Aventis/Regeneron) or is (or comprises) such antibody or a PCSK9-binding derivative thereof. In an example, the alirocumab is glycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos or Hek293 cell). Preferably, the ligand is alirocumab or SAR236553/REGN727.

In an embodiment, the ligand is or comprises the variable domains of antibody evolocumab or or is (or comprises) such antibody or a PCSK9-binding derivative thereof. In an example, the antibody is glycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos or Hek293 cell). Preferably, the ligand is evolocumab.

In an embodiment, the ligand is selected from evolocumab, 1D05-IgG2 (Merck & Co.), ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat) and alirocumab.

In an embodiment, the ligand is selected from the following (sequences and definitions as per US2011/0065902, incorporated herein by reference):—

1. An antibody or antigen-binding fragment thereof which specifically binds hPCSK9, wherein the antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair having SEQ ID NOs: 218/226. 2. The antibody or antigen-binding fragment of concept 1 comprising heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232. 3. The antibody or antigen-binding fragment of concept 2 comprising an HCVR having the amino acid sequence of SEQ ID NO: 218 and an LCVR having the amino acid sequence of SEQ ID NO: 226. 4. An antibody or antigen-binding fragment thereof which binds to the same epitope on hPCSK9 as an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232. 5. An antibody or antigen-binding fragment thereof which competes for binding to hPCSK9 with an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.

In an embodiment, the ligand is selected from the following (sequences and definitions as per US2012/0093818, incorporated herein by reference):—

1. An isolated neutralizing antigen binding protein that binds to a PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the neutralizing antigen binding protein decreases the LDLR lowering effect of PCSK9 on LDLR, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60. 2. The isolated neutralizing antigen binding protein of concept 2, wherein the antigen binding protein is a LDLR non-competitive neutralizing antigen binding protein. 3. The isolated neutralizing antigen binding protein of concept 2, wherein the antigen binding protein is a LDLR competitive neutralizing antigen binding protein. 4. An antigen binding protein that selectively binds to PCSK9, wherein said antigen binding protein binds to PCSK9 with a Kd that is less than 100 pM. 5. An antigen binding protein that binds to a PCSK 9 protein of SEQ ID NO: 303 in a first manner, wherein the antigen binding protein binds to a variant of PCSK9 in a second manner, wherein said PCSK9 variant has at least one point mutation at a position selected from the group consisting of: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 of SEQ ID NO: 303, wherein the first manner comprises a first EC50, a first Bmax, or a first EC50 and a first Bmax, wherein the second manner comprises a second EC50, a second Bmax, or a second EC50 and a second Bmax, and wherein a value for the first manner is different from a value for the second manner, and wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60. 6. The antigen binding protein of concept 6, wherein the first manner comprises a first Bmax, wherein the second manner comprises a second Bmax that is different from the first Bmax, and wherein said PCSK9 variant has at least one point mutation selected from the group consisting of: D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R. 7. The antigen binding protein of concept 6, wherein the antigen binding protein binds to PCSK9 at a location that overlaps with a location that LDLR binds to PCSK9. 8. A method of making an antigen binding protein that binds to a PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the antigen binding protein decreases the LDLR lowering effect of PCSK9 on LDLR, said method comprising: providing a host cell comprising a nucleic acid sequence that encodes the antigen binding protein; and maintaining the host cell under conditions in which the antigen binding protein is expressed, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60. 9. A method for treating or preventing a condition associated with elevated serum cholesterol levels in a subject, said method comprising administering to a subject in need thereof an effective amount of an isolated neutralizing antigen binding protein simultaneously or sequentially with an agent that elevates the availability of LDLR protein, wherein the isolated antigen binding protein binds to a PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the neutralizing antigen binding protein decreases the LDLR lowering effect of PCSK9 on LDLR, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60. 10. The method of concept 10, wherein the agent that elevates the availability of LDLR protein comprises a statin. 11. An antigen binding protein that binds to PCSK9, wherein when the antigen binding protein is bound to PCSK9, the antibody is positioned 8 angstroms or less from at least one of the following residues of PCSK9: S153, S188, 1189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154, T187, H193, E195, 1196, M201, V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372, C375, or C378, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.

The ligand can be used for the treatment, therapy, prophylaxis and/or diagnosis of one or more diseases or conditions or susceptibility thereto, wherein such diseases or conditions comprise those disclosed in US20120093818A1 (Amgen, Inc) and US20110065902A1 (Regeneron Pharmaceuticals, Inc), eg, a disease or condition disclosed in paragraphs [0375] to [0383] of US20120093818A1, which disclosure is incorporated herein by reference in its entirety for inclusion in one more claims herein.

The ligand can be administered to a human characterised as described in US20120093818A1 (Amgen, Inc) or US20110065902A1.

The ligand can be administered in a form or combination disclosed in US20120093818A1 (Amgen, Inc) or US20110065902A1, which disclosure is incorporated herein by reference. For example, the ligand with a drug, excipient, diluent or carrier as described in US20120093818A1 (Amgen, Inc) or US20110065902A1 (eg, as disclose in paragraphs [0384] to [0412] of US20120093818A1), which disclosure is incorporated herein by reference, and the present invention also relates to the corresponding pharmaceutical compositions comprising the combination of a ligand of the invention and such a further agent.

The ligand can be used in a method of diagnosis as set out in US20120093818A1 (Amgen, Inc) or US20110065902A1, eg, in paragraphs [0413] to [0415] of US20120093818A1 which disclosure is incorporated herein by reference.

Diagnostic Applications

In some embodiments, the ligand of the invention is a diagnostic tool. The ligand can be used to assay the amount of PCSK9 present in a sample and/or subject. As will be appreciated by one of skill in the art, such ligands need not be neutralizing ligands. In some embodiments, the diagnostic ligand is not a neutralizing ligand. In some embodiments, the diagnostic ligand binds to a different epitope than a neutralizing ligand binds to. In some embodiments, the two ligands do not compete with one another.

In some embodiments, the ligands of the invention are used or provided in an assay kit and/or method for the detection of PCSK9 in mammalian tissues or cells in order to screen/diagnose for a disease or disorder associated with changes in levels of PCSK9. The kit comprises a ligand that binds PCSK9 and means for indicating the binding of the ligand with PCSK9, if present, and optionally PCSK9 protein levels. Various means for indicating the presence of a ligand can be used. For example, fluorophores, other molecular probes, or enzymes can be linked to the ligand and the presence of the ligand can be observed in a variety of ways. The method for screening for such disorders can involve the use of the kit, or simply the use of one of the disclosed ligands and the determination of whether the ligand binds to PCSK9 in a sample. As will be appreciated by one of skill in the art, high or elevated levels of PCSK9 will result in larger amounts of the ligand binding to PCSK9 in the sample. Thus, degree of ligand binding can be used to determine how much PCSK9 is in a sample. Subjects or samples with an amount of PCSK9 that is greater than a predetermined amount (e.g., an amount or range that a person without a PCSK9 related disorder would have) can be characterized as having a PCSK9 mediated disorder. In some embodiments, the invention provides a method wherein the ligand is administered to a subject taking a statin, in order to determine if the statin has increased the amount of PCSK9 in the subject.

In some embodiments, the ligand is a non-neutralizing ligand and is used to determine the amount of PCSK9 in a subject receiving an ABP and/or statin treatment.

In some embodiments, the ligand of the invention can specifically bind human PCSK9 (eg, one, two or more rare variant forms disclosed herein) and is characterized by at least one of: (i) capable of reducing serum total cholesterol at least about 25-35% and sustaining the reduction over at least a 24 day period relative to a predose level; (ii) capable of reducing serum LDL cholesterol at least about 65-80% and sustaining the reduction over at least a 24 day period relative to a predose level; (iii) capable of reducing serum LDL cholesterol at least about 40-70% and sustaining the reduction over at least a 60 or 90 day period relative to a predose level; (iv) capable of reducing serum triglyceride at least about 25-40% relative to predose level; (v) does not reduce serum HDL cholesterol or reduces serum HDL cholesterol no more than 5% relative to predose level. In some embodiments, an isolated nucleic acid molecule is provided and it encodes the ligand. In some embodiments an expression vector is provided and comprises the nucleic acid molecule. In some embodiments, a pharmaceutical composition is provided and it can comprise the ligand and a pharmaceutically acceptable carrier. In some embodiments, a method is provided for treating a disease or condition which is ameliorated, improved, inhibited or prevented with a PCSK9 antagonist ligand of the invention. The method can comprise administering a therapeutic amount of the pharmaceutical composition or ligand to a subject in need thereof. In some embodiments, the subject is a human subject suffering from hypercholesterolemia, hyperlipidemia, indicated for LDL apheresis, identified as heterozygous for Familial Hypercholesterolemia, statin intolerant. statin uncontrolled, at risk for developing hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis and cardiovascular diseases. In some embodiments, a method of providing a treatment or therapy is provided to a subject. In some embodiments, the method comprises reducing serum cholesterol at least about 40-70% over at least 60 to 90 days. In some embodiments, a method of receiving treatment or therapy is provided, the method can comprise receiving a ligand thereof at a frequency of once every 60 to 90 days.

In one aspect, the invention provides a ligand of the invention which is or comprises an human antibody or antigen-binding fragment of a human antibody that specifically binds and inhibits human proprotein convertase subtilisin/kexin type 9 (hPCSK9, eg, one, two or more rare variant forms disclosed herein and optionally form a and/or form a′), characterized by the ability to reduce serum LDL cholesterol in a human by 40-80% over a 24, 60 or 90 day period relative to predose levels, with little or no reduction in serum HDL cholesterol and/or with little or no measurable effect on liver function, as determined by ALT and AST measurements.

In one embodiment, the ligand of the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds hPCSK9 and is characterized by at least one of:

(i) capable of reducing serum total cholesterol at least about 25-35% and sustaining the reduction over at least a 24 day period relative to a predose level, preferably the reduction in serum total cholesterol is at least about 30-40%; (ii) capable of reducing serum LDL cholesterol at least about 65-80% and sustaining the reduction over at least a 24 day period relative to a predose level; (iii) capable of reducing serum triglyceride at least about 25-40% relative to predose level; (iv) does not reduce serum HDL cholesterol or reduces serum HDL cholesterol no more than 5% relative to predose level.

See US2011/0065902 for definitions of these terms and optional features, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds hPCSK9 and is characterized by at least one of:

(i) capable of reducing serum LDL cholesterol at least about 40-70% and sustaining the reduction over at least a 60 or 90 day period relative to a predose level; (ii) capable of reducing serum triglyceride at least about 25-40% relative to predose level; (iii) does not reduce serum HDL cholesterol or reduces serum HDL cholesterol no more than 5% relative to predose level.

In one embodiment, the antibody or antigen-binding fragment is characterized as exhibiting an enhanced binding affinity (KD) for hPCSK9 at pH 5.5 relative to the KD at pH 7.4, as measured by plasmon surface resonance. In a specific embodiment, the antibody or fragment thereof exhibits at least a 20-fold, at least a 40-fold or at least a 50-fold enhanced affinity for PCSK9 at an acidic pH relative to a neutral pH, as measured by surface plasmon resonance.

In one embodiment, the antibody or antigen-binding fragment is characterized as not exhibiting an enhanced binding affinity for PCSK9 at an acidic pH relative to a neutral pH, as measured by surface plasmon resonance. In a specific embodiment, the antibody or fragment thereof exhibits a decreased binding affinity at an acidic pH.

In another embodiment, the antibody or antigen-binding fragment binds human, human GOF mutation D374Y, cynomolgus monkey, rhesus monkey, mouse, rat and hamster PCSK9.

In one embodiment, the antibody or antigen-binding fragment binds human and monkey PCSK9, but does not bind mouse, rat or hamster PCSK9.

In one embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody comprising one or more of a heavy chain variable region (HCVR), light chain variable region (LCVR), HCDR1, HCDR2, HCDR3 disclosed in any of paragraphs [023]-[037] of US2011/0065902, the disclosures of which are incorporated herein by reference.

In a related embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody which specifically binds hPCSK9, wherein the antibody or fragment comprises heavy and light chain CDR domains contained within heavy and light chain sequence pairs selected from the group consisting of SEQ ID NO (using the sequence numbering in US2011/0065902): 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/514, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706, 714/716, 718/720, 722/730, 738/740 and 742/744. In one embodiment, the CDR sequences are contained within HCVR and LCVR selected from the amino acid sequence pairs of SEQ ID NO: 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 122/130, 138/140, 142/144, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 314/322, 330/332 and 334/336. In more specific embodiments, the CDR sequences are comprised within HCVR/LCVR sequences selected from SEQ ID NO: 90/92 or 218/226.

In an example, the invention features a pharmaceutical composition comprising a ligand of the invention, wherein the ligand comprises or consists of a recombinant human antibody or fragment thereof which specifically binds hPCSK9 and a pharmaceutically acceptable carrier. In one embodiment, the invention features a composition which is a combination of a ligand of the invention (eg, an antibody or antigen-binding fragment of an antibody), and a second therapeutic agent. The second therapeutic agent may be any agent that is advantageously combined with the ligand of the invention, for example, an agent capable of inducing a cellular depletion of cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase, such as, for example, cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc; capable of inhibiting cholesterol uptake and or bile acid re-absorption; capable of increasing lipoprotein catabolism (such as niacin); and/or activators of the LXR transcription factor that plays a role in cholesterol elimination such as 22-hydroxycholesterol.

In an example, the invention provides a method for inhibiting hPCSK9 activity using the anti-PCSK9 ligand of the invention (eg, an antibody or antigen-binding portion of the antibody of the invention), wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the invention. The disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of PCSK9 activity. Specific populations treatable by the therapeutic methods of the invention include subjects indicated for LDL apheresis, subjects with PCSK9-activating mutations (gain of function mutations, “GOF”), subjects with heterozygous Familial Hypercholesterolemia (heFH); subjects with primary hypercholesterolemia who are statin intolerant or statin uncontrolled; and subjects at risk for developing hypercholesterolemia who may be preventably treated. Other indications include dyslipidemia associated with secondary causes such as Type 2 diabetes mellitus, cholestatic liver diseases (primary biliary cirrhosis), nephrotic syndrome, hypothyroidism, obesity; and the prevention and treatment of atherosclerosis and cardiovascular diseases.

In specific embodiments of the method of the invention, the ligand of the invention (eg, anti-hPCSK9 antibody or antibody fragment of the invention) is useful to reduce elevated total cholesterol, non-HDL cholesterol, LDL cholesterol, and/or apolipoprotein B (apolipoprotein B100).

The ligand (eg, antibody or antigen-binding fragment) of the invention may be used alone or in combination with a second agent, for example, an HMG-CoA reductase inhibitor and/or another lipid lowering drug.

Treatment Population

The invention provides therapeutic methods for treating a human patient in need of a composition or ligand of the invention. While modifications in lifestyle and conventional drug treatment are often successful in reducing cholesterol levels, not all patients are able to achieve the recommended target cholesterol levels with such approaches. Various conditions, such as familial hypercholesterolemia (FH), appear to be resistant to lowering of LDL-C levels in spite of aggressive use of conventional therapy. Homozygous and heterozygous familial hypercholesterolemia (hoFH, heFH) is a condition associated with premature atherosclerotic vascular disease. However, patients diagnosed with hoFH are largely unresponsive to conventional drug therapy and have limited treatment options. Specifically, treatment with statins, which reduce LDL-C by inhibiting cholesterol synthesis and upregulating the hepatic LDL receptor, may have little effect in patients whose LDL receptors are non-existent or defective. A mean LDL-C reduction of only less than about 20% has been recently reported in patients with genotype-confirmed hoFH treated with the maximal dose of statins. The addition of ezetimibe 10 mg/day to this regimen resulted in a total reduction of LDL-C levels of 27%, which is still far from optimal. Likewise, many patients are statin non-responsive, poorly controlled with statin therapy, or cannot tolerate statin therapy; in general, these patients are unable to achieve cholesterol control with alternative treatments. There is a large unmet medical need for new treatments that can address the short-comings of current treatment options.

Specific populations treatable by the therapeutic methods of the invention include patients indicated for LDL apheresis, subjects with PCSK9-activating (GOF) mutations, heterozygous Familial Hypercholesterolemia (heFH); subjects with primary hypercholesterolemia who are statin intolerant or statin uncontrolled; and subjects at risk for developing hypercholesterolemia who may be preventably treated.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the anti-PCSK9 ligands, antibodies or antigen-binding fragments thereof of the present invention. The administration of therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When the ligand, eg, antibody, of the present invention is used for treating various conditions and diseases associated with PCSK9, including hypercholesterolemia, disorders associated with LDL and apolipoprotein B, and lipid metabolism disorders, and the like, in an adult patient, it is advantageous to intravenously administer the ligand or antibody of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, thus the composition invention provides the ligand by e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule. A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

The invention provides therapeutic methods in which the ligand, eg, antibody or antibody fragment, of the invention is useful to treat hypercholesterolemia associated with a variety of conditions involving hPCSK9. The anti-PCSK9 ligands, eg, antibodies or antibody fragments, of the invention are particularly useful for the treatment of hypercholesterolemia and the like. Combination therapies may include the anti-PCSK9 ligand of the invention with, for example, one or more of any agent that (1) induces a cellular depletion of cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase, such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin; (2) inhibits cholesterol uptake and or bile acid re-absorption; (3) increase lipoprotein catabolism (such as niacin); and activators of the LXR transcription factor that plays a role in cholesterol elimination such as 22-hydroxycholesterol or fixed combinations such as ezetimibe plus simvastatin; a statin with a bile resin (e.g., cholestyramine, colestipol, colesevelam), a fixed combination of niacin plus a statin (e.g., niacin with lovastatin); or with other lipid lowering agents such as omega-3-fatty acid ethyl esters (for example, omacor).

Tailoring Antibodies to Rare PCSK9 Variant Profile

As outline above, the invention includes the possibility to tailor treatment of humans further by selecting antibody-based ligands with variable domains based on gene segments commonly found in humans of the ethnic populations where the variant PCSK9 forms are found to meet the selection criteria of the invention. An example is provided below for ligands comprising antibody VH domains derived from recombination of human VH3-23.

The inventor analysed the frequencies and distribution of various human VH3-23 alleles and realised the desirability of using ligands based on human VH3-23 alleles comprising SNP rs56069819. This SNP corresponds to a change from leucine at position 24 in the encoded protein sequence to a valine at that position (L24V change) and the SNP is at coordinate 106268889 on human chromosome 14.

FIG. 2 shows the cumulative allele frequency distribution across the 1000 Genomes Project database of human VH3-23 alleles comprising SNP rs56069819 (such alleles denoted “C” and the most frequent allele (which does not comprise this SNP) denoted “A”). The figure shows that VH3-23 alleles comprising SNP rs56069819 are present at a cumulative frequency of 11% across all human ethnic populations taken as a whole, whereas in certain specific human ethnic sub-populations (ASW, LWK, YRI, CEU and GBR) such alleles are present at an above-average cumulative frequency. Indicated in the figure are those human PCSK9 variant forms (marked “Variants”) that are found in the various sub-populations with above-average occurrence of human VH3-23 alleles comprising SNP rs56069819. Table 7 shows the VH3-23 variants and the SNPs that they comprise, as well as their cumulative allele frequencies as found in the 1000 Genomes Project database.

Notably, human VH3-23 alleles comprising SNP rs56069819 were found in the CEU population at a frequency that is almost double the frequency of 11% for all populations. For the ASW and YRI populations the frequency was over a quarter of the population. Thus, the invention advantageously enables one to select a ligand comprising an antibody or antibody fragment, wherein the antibody or fragment comprises a VH domain derived from the recombination of a human VH gene segment, a human D gene segment and a human JH gene segment, the VH gene segment comprising a nucleotide sequence that comprises SNP rs56069819 (dbSNP numbering, build number as recited above).

In an example, one can tailor the treatment further by selecting such a ligand that specifically binds to a human PCSK9 selected from forms: f, c, m, e, h, p, q and aj, such forms being those appearing in human populations ASW, LWK, YRI, CEU and GBR.

In an example, the ligand specifically binds to a human PCSK9 comprising a mutation I474V in SEQ ID NO: 1, wherein the human is of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises gene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example, additionally the ligand is an antibody or fragment comprising a VH domain derived from the recombination of human gene segment VH3-23*04.

In an example, the ligand specifically binds to a human PCSK9 comprising a mutation I474V in SEQ ID NO: 1 (eg, form f r, p, e or aj), wherein the human is of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises gene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example, additionally the ligand is an antibody or fragment comprising a VH domain derived from the recombination of human gene segment VH3-23*04. Optionally, the ligand is for treating or preventing dislipidemia in said human, eg, for reducing cholesterol or maintaining a previously reduced cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprising a mutation E670G in SEQ ID NO: 1 (eg, form c, r or q), wherein the human is of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises gene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example, additionally the ligand is an antibody or fragment comprising a VH domain derived from the recombination of human gene segment VH3-23*04. Optionally, the ligand is for treating or preventing dislipidemia in said human, eg, for reducing cholesterol or maintaining a previously reduced cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprising a mutation Q619P in SEQ ID NO: 1 (eg, form h), wherein the human is of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises gene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example, additionally the ligand is an antibody or fragment comprising a VH domain derived from the recombination of human gene segment VH3-23*04. Optionally, the ligand is for treating or preventing dislipidemia in said human, eg, for reducing cholesterol or maintaining a previously reduced cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprising a mutation N425S in SEQ ID NO: 1 (eg, form e), wherein the human is of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises gene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example, additionally the ligand is an antibody or fragment comprising a VH domain derived from the recombination of human gene segment VH3-23*04. Optionally, the ligand is for treating or preventing dislipidemia in said human, eg, for reducing cholesterol or maintaining a previously reduced cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprising a mutation R46L in SEQ ID NO: 1 (eg, form aj), wherein the human is of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises gene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example, additionally the ligand is an antibody or fragment comprising a VH domain derived from the recombination of human gene segment VH3-23*04. Optionally, the ligand is for treating or preventing dislipidemia in said human, eg, for increasing cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprising a mutation A53V in SEQ ID NO: 1 (eg, form p or q), wherein the human is of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises gene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example, additionally the ligand is an antibody or fragment comprising a VH domain derived from the recombination of human gene segment VH3-23*04. Optionally, the ligand is for treating or preventing dislipidemia in said human, eg, for increasing cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprising a mutation A443T in SEQ ID NO: 1 (eg, form m or h), wherein the human is of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises gene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example, additionally the ligand is an antibody or fragment comprising a VH domain derived from the recombination of human gene segment VH3-23*04. Optionally, the ligand is for treating or preventing dislipidemia in said human, eg, for increasing cholesterol level in said human.

In an example, the VH gene segment is VH3-23*04, which is a commonly found variant that comprises SNP rs56069819 in human populations ASW, LWK, YRI, CEU and GBR.

In an example, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human that expresses a human PCSK9 selected from forms: f, c, m, e, h, p, q and aj.

In an example, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of ASW, LWK, YRI, CEU or GBR ancestry.

In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of ASW ancestry, wherein the human expresses a PCSK9 selected from f, c, m, e, h, p and q or the human comprises a corresponding nucleotide or amino acid sequence as set out in Table 6. Optionally this ligand comprises a VH domain derived from recombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of LWK ancestry, wherein the human expresses a PCSK9 selected from f, c, m, e and h or the human comprises a corresponding nucleotide or amino acid sequence as set out in Table 6. Optionally this ligand comprises a VH domain derived from recombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of YRI ancestry, wherein the human expresses a PCSK9 selected from f, c, m, e and h or the human comprises a corresponding nucleotide or amino acid sequence as set out in Table 6. Optionally this ligand comprises a VH domain derived from recombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of CEU ancestry, wherein the human expresses a PCSK9 selected from f, c, p and aj or the human comprises a corresponding nucleotide or amino acid sequence as set out in Table 6. Optionally this ligand comprises a VH domain derived from recombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of GBR ancestry, wherein the human expresses a PCSK9 selected from f c and p or the human comprises a corresponding nucleotide or amino acid sequence as set out in Table 6. Optionally this ligand comprises a VH domain derived from recombination of human VH3-23*04.

In an example, the ligand is alirocumab.

In other embodiments of any configuration or TOI herein, as explained more fully above, the invention provides for ligands which are tailored to the human recipient's genotype and/or phenotype based on alternative human VH gene segments, or on Vκ, Vλ or constant region gene segments (see further Table 9 for representative variants).

For example, the ligand of the invention comprises or consists of an antibody that comprises a VH domain that is derived from the recombination of a human VH gene segment, a human D gene segment and a human JH gene segment, wherein the VH gene segment is selected from the group consisting of (i) IGHV1-18*01 and the genome of the human comprises a human IGHV1-18*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGHV1-18*01; or (ii) IGVH1-46*01 and the genome of the human comprises a human IGHV1-46*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGHV1-46*01.

For example, the ligand of the invention comprises or consists of an antibody that comprises a VL domain that is derived from the recombination of a human VL gene segment and a human JL gene segment, wherein the VL gene segment is selected from the group consisting of (i) IGKV4-1*01 and the genome of the human comprises a human IGKV4-1*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGKV4-1*01; (ii) IGLV2-14*01 and the genome of the human comprises a human IGLV2-14*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGLV2-14*01; or (iii) IGKV1-13*02 and the genome of the human comprises a human IGKV1-13*02 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGKV1-13*02.

For example, the inventor identified the possibility of addressing the rarer IGH-gamma-1 SNPs 204D (observed cumulative frequency of 0.296) and 206L (observed cumulative frequency of 0.283) individually or in combination. These residues are part of the CH3 domain, and as such they form part of antibody Fc regions. Thus, matching of these CH3 variations with the patient is especially beneficial for reasons as discussed above. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein the genome of the human comprises a gamma-1 heavy chain constant region nucleotide sequence that encodes such an Asp or Leu or the human expresses antibodies comprising human gamma-1 constant regions comprising such an Asp or Leu. An example of such a ligand is alirocumab.

In another example, the inventor identified the possibility of addressing IGH-gamma-2 SNPs. This included consideration of Fc region variation—in this respect, the inventor focused on positions 161 and 257 which are in the Fc region. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44; and wherein the genome of the human comprises a gamma-2 heavy chain constant region nucleotide sequence that encodes such a selected amino acid or the human expresses antibodies comprising human gamma-2 constant regions comprising such a selected amino acid. An example of such a ligand is evolocumab or bococizumab.

In another example, the inventor addressed human kappa constant region variation. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50; and wherein the genome of the human comprises a kappa light chain constant region nucleotide sequence that encodes such a Val or Cys or the human expresses antibodies comprising human kappa light chain constant regions comprising such a Val or Cys. An example of such a ligand is alirocumab or bococizumab.

In another example, the inventor addressed human lambda constant region variation. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human IGLC2*01 light chain constant region; and wherein the genome of the human comprises a human IGLC2*01 nucleotide sequence or the human expresses antibodies comprising human light chain IGLC2*01 constant regions. An example of such a ligand is evolocumab.

Further exemplary ligands are in the paragraphs 1-17 as follows.

-   -   1. An antibody or antibody fragment for use in a method of (a)         reducing cholesterol level or maintaining previously reduced         cholesterol level in a human in need thereof, or (b) targeting         PCSK9 in a human,         -   wherein the antibody or fragment comprises a human gamma             heavy chain constant region that comprises a first amino             acid that is encoded by a human gamma heavy chain constant             region gene segment SNP, and the antibody or fragment             specifically binds a proprotein convertase subtilisin/kexin             type 9 (PCSK9) amino acid sequence that comprises a             C-terminal domain comprising a mutation selected from the             group consisting of I474V, Q619P, N425S and E670G (eg,             selected from I474V and E670G) in SEQ ID NO: 1, wherein the             human comprises a nucleotide sequence encoding said PCSK9             amino acid sequence and comprises a human gamma heavy chain             constant region gene segment comprising said SNP, or the             human expresses antibodies comprising human gamma constant             regions comprising said first amino acid.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

In an alternative, paragraph 1 provides:—

An antibody or antibody fragment for use in a method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, or (b) targeting PCSK9 in a human,

wherein the antibody or fragment comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO:1, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 constant regions comprising such an Asp and Leu.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G. In another embodiment, said mutation is N425S. In another embodiment, said mutation is Q619P.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

An example provides:—

An antibody or antibody fragment for use in a method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, or (b) targeting PCSK9 in a human,

wherein the antibody or fragment comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V in SEQ ID NO:1, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGHG1*01 human heavy chain constant region gene segment. Optionally, the antibody or fragment comprises an IGHG1*01 human heavy chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

Another example provides:—

An antibody or antibody fragment for use in a method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, or (b) targeting PCSK9 in a human,

wherein the antibody or fragment comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation E670G in SEQ ID NO:1, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGHG1*01 human heavy chain constant region gene segment. Optionally, the antibody or fragment comprises an IGHG1*01 human heavy chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

In an example, said antibody or antibody fragment has been determined to specifically bind a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation selected from the group consisting of I474V, Q619P, N425S and E670G (eg, selected from I474V and E670G) in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1.

-   -   2. The antibody or antibody fragment of paragraph 1, wherein the         antibody comprises a human gamma-1 heavy chain constant region         that comprises an Asp corresponding to position 204 of SEQ ID         NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42.     -   3. The antibody or antibody fragment of paragraph 1 or 2,         wherein the antibody comprises an IGHG1*01 human heavy chain         constant region.     -   4. The antibody or antibody fragment of any one of paragraphs 1         to 3, wherein the human has been determined to comprise the         nucleotide sequence that encodes a PCSK9 comprising a C-terminal         domain comprising said mutation (eg, I474V or E670G) in SEQ ID         NO: 1 and/or a proprotein convertase subtilisin/kexin type 9         (PCSK9) variant protein encoded by the nucleotide sequence of         SEQ ID NO: 29 or 30.     -   5. The antibody or antibody fragment of any one of paragraphs 1         to 4, the method comprising the step of determining that the         human comprises the nucleotide sequence that encodes a PCSK9         comprising a C-terminal domain comprising said mutation (eg,         I474V or E670G) and/or a proprotein convertase subtilisin/kexin         type 9 (PCSK9) variant protein comprising said mutation (eg,         I474V or E670G), optionally, wherein the determining step is         performed before administration of the antibody to the human.     -   6. The antibody or antibody fragment of paragraph 5, wherein the         step of determining comprises assaying a biological sample from         the human for a nucleotide sequence encoding the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1.     -   7. The antibody or antibody fragment of paragraph 6, wherein the         assaying comprises contacting the biological sample with         -   a. at least one oligonucleotide probe comprising a sequence             of at least 10 contiguous nucleotides that can specifically             hybridize to and identify in the biological sample a             nucleotide sequence encoding the PCSK9 that comprises the             C-terminal domain comprising the mutation (eg, I474V or             E670G) in SEQ ID NO: 1 or that specifically hybridizes to an             antisense of said sequence, wherein said nucleic acid             hybridizes to at least one nucleotide present in said             selected sequence which is not present in SEQ ID NO: 28 or             hybridizes to an antisense sequence thereby forming a             complex when at least one nucleotide sequence encoding the             PCSK9 that comprises the C-terminal domain comprising the             mutation (eg, I474V or E670G) in SEQ ID NO: 1 is present;             and/or         -   b. at least one oligonucleotide probe comprising a sequence             of at least 10 contiguous nucleotides of a nucleotide             sequence encoding the PCSK9 that comprises the C-terminal             domain comprising the mutation (eg, I474V or E670G) in SEQ             ID NO: 1 or comprising an antisense sequence of said             contiguous nucleotides, wherein said sequence of contiguous             nucleotides comprises at least one nucleotide present in             said selected sequence which is not present in SEQ ID NO: 28             thereby forming a complex when the nucleotide sequence             encoding the PCSK9 that comprises a C-terminal domain             comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1             is present; and detecting the presence or absence of the             complex, wherein detecting the presence of the complex             determines that the human comprises the PCSK9 that comprises             the C-terminal domain comprising the mutation (eg, I474V or             E670G) in SEQ ID NO: 1.     -   8. The antibody or antibody fragment of paragraph 6 or 7,         wherein the assaying comprises nucleic acid amplification and         optionally one or more methods selected from sequencing, next         generation sequencing, nucleic acid hybridization, and         allele-specific amplification and/or wherein the assaying is         performed in a multiplex format.     -   9. The antibody or antibody fragment of any one of paragraphs 1         to 8, wherein said antibody or antibody fragment is for         administration to a human that is or has been further determined         to be substantially resistant to statin treatment.     -   10. The antibody or antibody fragment of any one of paragraphs 1         to 9, wherein antibody or antibody fragment is for         administration to a human that is receiving or has received         statin treatment or has reduced responsiveness to statin         treatment.     -   11. The antibody or antibody fragment of paragraph 9 or 10,         wherein said antibody or antibody fragment is for administration         to the human separately or simultaneously with said statin         treatment.     -   12. The antibody or antibody fragment of any one of paragraphs 6         to 10, wherein said biological sample comprises serum, blood,         faeces, tissue, a cell, urine and/or saliva of said human.     -   13. The antibody or antibody fragment of any one of paragraphs 1         to 12, wherein said human is indicated as heterozygous for a         nucleotide sequence encoding the PCSK9 C-terminal domain         comprising a said PCSK9 mutation (eg, I474V or E670G),         optionally, wherein said human is further indicated as         comprising the nucleotide sequence of SEQ ID NO: 28, or said         human is indicated as homozygous for a nucleotide sequence         encoding the PCSK9 C-terminal domain comprising a said PCSK9         mutation (eg, I474V or E670G) in SEQ ID NO: 1.     -   14. The antibody or antibody fragment of any one of paragraphs 1         to 13, wherein said human has been diagnosed with at least one         condition selected from a lipid disorder, hyperlipoproteinemia,         hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart         attack, a stroke, coronary heart disease, atherosclerosis,         peripheral vascular disease, claudication (eg, claudication         associated with elevated cholesterol) and high blood pressure.     -   15. The antibody or antibody fragment of any one of paragraphs 1         to 14, wherein said antibody or antibody fragment treats or         reduces the risk in said human of at least one condition         selected from a lipid disorder, hyperlipoproteinemia,         hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart         attack, a stroke, coronary heart disease, atherosclerosis,         peripheral vascular disease, claudication (eg, claudication         associated with elevated cholesterol) and high blood pressure.     -   16. The antibody or antibody fragment of any one of paragraphs 1         to 15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.     -   17. The antibody or antibody fragment of any one of paragraphs 1         to 16, wherein said antibody or antibody fragment is for         administration by intravenous or subcutaneous administration         and/or is comprised in an injectable preparation.

Further exemplary methods are in the paragraphs 1-18 as follows.

-   1. A method of (a) reducing cholesterol level or maintaining     previously reduced cholesterol level in a human in need thereof     or (b) targeting PCSK9 in a human, the method comprising     administering to said human an antibody or antibody fragment that     specifically binds a proprotein convertase subtilisin/kexin type 9     (PCSK9) that comprises a C-terminal domain comprising a said PCSK9     mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody     or fragment comprises a human gamma-1 heavy chain constant region     that comprises an Asp corresponding to position 204 of SEQ ID NO: 42     or a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein     said human comprises (i) an IGHG1*01 human heavy chain constant     region gene segment, or the human expresses antibodies comprising     human gamma-1 heavy chain constant regions comprising such an Asp     and Leu and (ii) a nucleotide sequence encoding said proprotein     convertase subtilisin/kexin type 9 (PCSK9) that comprises a     C-terminal domain comprising said mutation (eg, I474V or E670G) in     SEQ ID NO: 1.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

An example provides:—

A method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation 1474 in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation I474V in SEQ ID NO: 1. Optionally, the antibody or fragment comprises a IGHG1*01 human heavy chain constant region. In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

Another example provides:—

A method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation E670G in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation E670G in SEQ ID NO: 1. Optionally, the antibody or fragment comprises an IGHG1*01 human heavy chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

In an example, said antibody or antibody fragment has been determined to specifically bind a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1.

-   2. The method of paragraph 1, comprising, before said administering,     selecting a human comprising said nucleotide sequence of (ii),     wherein the human is the human of paragraph 1. -   3. The method of paragraph 1 or 2, wherein the antibody comprises a     human gamma-1 heavy chain constant region that comprises an Asp     corresponding to position 204 of SEQ ID NO: 42 and a Leu     corresponding to position 206 of SEQ ID NO: 42. -   4. The method of paragraph 1, 2 or 3, wherein the antibody comprises     an IGHG1*01 human heavy chain constant region. -   5. The method of any one of paragraphs 1 to 4, wherein the human has     been determined to comprise the nucleotide sequence that encodes a     PCSK9 comprising a C-terminal domain comprising said mutation (eg,     I474V or E670G) in SEQ ID NO: 1 and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein encoded by the     nucleotide sequence of SEQ ID NO: 29 or 30. -   6. The method of any one of paragraphs 1 to 5, comprising the step     of determining that the human comprises the nucleotide sequence that     encodes a PCSK9 comprising a C-terminal domain comprising said     mutation (eg, I474V or E670G) and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein comprising said     mutation (eg, I474V or E670G), optionally, wherein the determining     step is performed before administration of the antibody to the     human. -   7. The method of paragraph 6, wherein the step of determining     comprises assaying a biological sample from the human for a     nucleotide sequence encoding the PCSK9 that comprises the C-terminal     domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1. -   8. The method of paragraph 7, wherein the assaying comprises     contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   9. The method of paragraph 7 or 8, wherein the assaying comprises     nucleic acid amplification and optionally one or more methods     selected from sequencing, next generation sequencing, nucleic acid     hybridization, and allele-specific amplification and/or wherein the     assaying is performed in a multiplex format. -   10. The method of any one of paragraphs 1 to 9, wherein said human     is or has been further determined to be substantially resistant to     statin treatment. -   11. The method of any one of paragraphs 1 to 10, wherein said human     is receiving or has received statin treatment or has reduced     responsiveness to statin treatment. -   12. The method of paragraph 10 or 11, wherein said antibody or     antibody fragment is administered to the human separately or     simultaneously with said statin treatment. -   13. The method of any one of paragraphs 7 to 9, wherein said     biological sample comprises serum, blood, faeces, tissue, a cell,     urine and/or saliva of said human. -   14. The method of any one of paragraphs 1 to 13, wherein said human     is indicated as heterozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation (eg, I474V or     E670G), optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 28, or said human     is indicated as homozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation I474V or E670G in     SEQ ID NO: 1. -   15. The method of any one of paragraphs 1 to 14, wherein said human     has been diagnosed with at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   16. The method of any one of paragraphs 1 to 15, wherein said     antibody or antibody fragment treats or reduces the risk in said     human of at least one condition selected from a lipid disorder,     hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   17. The method of any one of paragraphs 1 to 16, wherein the     nucleotide sequence is SEQ ID NO: 29 or 30. -   18. The method of any one of paragraphs 1 to 17, wherein said     antibody or antibody fragment is administered by intravenous or     subcutaneous administration and/or is comprised in an injectable     preparation.

Further exemplary ligands are in the paragraphs 1-17 as follows.

-   1. An antibody or antibody fragment for use in a method of (a)     reducing cholesterol level or maintaining previously reduced     cholesterol level in a human in need thereof or (b) targeting PCSK9     in a human,     -   wherein the antibody comprises a human gamma-2 heavy chain         constant region that comprises an amino acid selected from the         group consisting of a Pro corresponding to position 72 of SEQ ID         NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a         Phe corresponding to position 76 of SEQ ID NO: 44, a Val         corresponding to position 161 of SEQ ID NO: 44 and an Ala         corresponding to position 257 of SEQ ID NO: 44, and the antibody         or fragment specifically binds a proprotein convertase         subtilisin/kexin type 9 (PCSK9) amino acid sequence that         comprises a C-terminal domain comprising a said PCSK9 mutation         (eg, I474V or E670G) in SEQ ID NO:1, wherein the human comprises         a nucleotide sequence encoding said amino acid sequence and         comprises an IGHG2*01 human heavy chain constant region gene         segment, or the human expresses antibodies comprising human         gamma-2 constant regions comprising such a Pro, Asn, Phe, Val         and Ala.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

An example provides:—

An antibody or antibody fragment for use in a method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

wherein the antibody comprises a human gamma-2 heavy chain constant region that comprises a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V in SEQ ID NO:1, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGHG2*01 human heavy chain constant region gene segment. Optionally, the antibody or fragment comprises an IGHG2*01 human heavy chain constant

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human. region.

Another example provides:—

An antibody or antibody fragment for use in a method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

wherein the antibody comprises a human gamma-2 heavy chain constant region that comprises a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation E670G in SEQ ID NO:1, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGHG2*01 human heavy chain constant region gene segment. Optionally, the antibody or fragment comprises an IGHG2*01 human heavy chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

In an example, said antibody or antibody fragment has been determined to specifically bind a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-2 heavy chain constant region that comprises a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1.

-   2. The antibody or antibody fragment of paragraph 1, wherein the     antibody comprises a human gamma-2 heavy chain constant region that     comprises a Pro corresponding to position 72 of SEQ ID NO: 44, an     Asn corresponding to position 75 of SEQ ID NO: 44, a Phe     corresponding to position 76 of SEQ ID NO: 44, a Val corresponding     to position 161 of SEQ ID NO: 44 and an Ala corresponding to     position 257 of SEQ ID NO: 44. -   3. The antibody or antibody fragment of paragraph 1 or 2, wherein     the antibody comprises an IGHG2*01 human heavy chain constant     region. -   4. The antibody or antibody fragment of any one of paragraphs 1 to     3, wherein the human has been determined to comprise the nucleotide     sequence that encodes a PCSK9 comprising a C-terminal domain     comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1 and/or     a proprotein convertase subtilisin/kexin type 9 (PCSK9) variant     protein encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. -   5. The antibody or antibody fragment of any one of paragraphs 1 to     4, the method comprising the step of determining that the human     comprises the nucleotide sequence that encodes a PCSK9 comprising a     C-terminal domain comprising said mutation (eg, I474V or E670G)     and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)     variant protein comprising said mutation (eg, I474V or E670G),     optionally, wherein the determining step is performed before     administration of the antibody to the human. -   6. The antibody or antibody fragment of paragraph 5, wherein the     step of determining comprises assaying a biological sample from the     human for a nucleotide sequence encoding the PCSK9 that comprises     the C-terminal domain comprising the mutation (eg, I474V or E670G)     in SEQ ID NO: 1. -   7. The antibody or antibody fragment of paragraph 6, wherein the     assaying comprises contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   8. The antibody or antibody fragment of paragraph 6 or 7, wherein     the assaying comprises nucleic acid amplification and optionally one     or more methods selected from sequencing, next generation     sequencing, nucleic acid hybridization, and allele-specific     amplification and/or wherein the assaying is performed in a     multiplex format. -   9. The antibody or antibody fragment of any one of paragraphs 1 to     8, wherein said antibody or antibody fragment is for administration     to a human that is or has been further determined to be     substantially resistant to statin treatment. -   10. The antibody or antibody fragment of any one of paragraphs 1 to     9, wherein antibody or antibody fragment is for administration to a     human that is receiving or has received statin treatment or has     reduced responsiveness to statin treatment. -   11. The antibody or antibody fragment of paragraph 9 or 10, wherein     said antibody or antibody fragment is for administration to the     human separately or simultaneously with said statin treatment. -   12. The antibody or antibody fragment of any one of paragraphs 6 to     8, wherein said biological sample comprises serum, blood, faeces,     tissue, a cell, urine and/or saliva of said human. -   13. The antibody or antibody fragment of any one of paragraphs 1 to     12, wherein said human is indicated as heterozygous for a nucleotide     sequence encoding the PCSK9 C-terminal domain comprising a said     PCSK9 mutation (eg, I474V or E670G), optionally, wherein said human     is further indicated as comprising the nucleotide sequence of SEQ ID     NO: 28, or said human is indicated as homozygous for a nucleotide     sequence encoding the PCSK9 C-terminal domain comprising a mutation     I474V or E670G in SEQ ID NO: 1. -   14. The antibody or antibody fragment of any one of paragraphs 1 to     13, wherein said human has been diagnosed with at least one     condition selected from a lipid disorder, hyperlipoproteinemia,     hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,     a stroke, coronary heart disease, atherosclerosis, peripheral     vascular disease, claudication (eg, claudication associated with     elevated cholesterol) and high blood pressure. -   15. The antibody or antibody fragment of any one of paragraphs 1 to     14, wherein said antibody or antibody fragment treats or reduces the     risk in said human of at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   16. The antibody or antibody fragment of any one of paragraphs 1 to     15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30. -   17. The antibody or antibody fragment of any one of paragraphs 1 to     16, wherein said antibody or antibody fragment is for administration     by intravenous or subcutaneous administration and/or is comprised in     an injectable preparation.

Further exemplary methods are in the paragraphs 1-18 as follows.

-   1. A method of (a) reducing cholesterol level or maintaining     previously reduced cholesterol level in a human in need thereof     or (b) targeting PCSK9 in a human, the method comprising     administering to said human an antibody or antibody fragment that     specifically binds a proprotein convertase subtilisin/kexin type 9     (PCSK9) that comprises a C-terminal domain comprising a said PCSK9     mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody     or fragment comprises a human gamma-2 heavy chain constant region     that comprises an amino acid selected from the group consisting of a     Pro corresponding to position 72 of SEQ ID NO: 44, an Asn     corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding     to position 76 of SEQ ID NO: 44, a Val corresponding to position 161     of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID     NO: 44 and wherein said human comprises (i) an IGHG2*01 human heavy     chain constant region gene segment, or the human expresses     antibodies comprising human gamma-2 heavy chain constant regions     comprising such a Pro, Asn, Phe, Val and Ala and (ii) a nucleotide     sequence encoding said proprotein convertase subtilisin/kexin type 9     (PCSK9) that comprises a C-terminal domain comprising said mutation     (eg, I474V or E670G) in SEQ ID NO: 1.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

An example provides:—

A method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human, the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation 1474 in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-2 heavy chain constant region that comprises a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation I474V in SEQ ID NO: 1. Optionally, the antibody or fragment comprises an IGHG2*01 human heavy chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

Another example provides:—

A method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation E670G in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-2 heavy chain constant region that comprises a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation E670G in SEQ ID NO: 1. Optionally, the antibody or fragment comprises an IGHG2*01 human heavy chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

In an example, said antibody or antibody fragment has been determined to specifically bind a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-2 heavy chain constant region that comprises a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1.

-   2. The method of paragraph 1, comprising, before said administering,     selecting said human comprising said nucleotide sequence of (ii). -   3. The method of paragraph 1 or 2, wherein the antibody comprises a     human gamma-1 heavy chain constant region that comprises a Pro     corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding     to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76     of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO:     44 and an Ala corresponding to position 257 of SEQ ID NO: 44. -   4. The method of paragraph 1, 2 or 3, wherein the antibody comprises     an IGHG2*01 human heavy chain constant region. -   5. The method of any one of paragraphs 1 to 4, wherein the human has     been determined to comprise the nucleotide sequence that encodes a     PCSK9 comprising a C-terminal domain comprising said mutation (eg,     I474V or E670G) in SEQ ID NO: 1 and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein encoded by the     nucleotide sequence of SEQ ID NO: 29 or 30. -   6. The method of any one of paragraphs 1 to 5, comprising the step     of determining that the human comprises the nucleotide sequence that     encodes a PCSK9 comprising a C-terminal domain comprising said     mutation (eg, I474V or E670G) and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein comprising said     mutation (eg, I474V or E670G), optionally, wherein the determining     step is performed before administration of the antibody to the     human. -   7. The method of paragraph 6, wherein the step of determining     comprises assaying a biological sample from the human for a     nucleotide sequence encoding the PCSK9 that comprises the C-terminal     domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1. -   8. The method of paragraph 7, wherein the assaying comprises     contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   9. The method of paragraph 7 or 8, wherein the assaying comprises     nucleic acid amplification and optionally one or more methods     selected from sequencing, next generation sequencing, nucleic acid     hybridization, and allele-specific amplification and/or wherein the     assaying is performed in a multiplex format. -   10. The method of any one of paragraphs 1 to 9, wherein said human     is or has been further determined to be substantially resistant to     statin treatment. -   11. The method of any one of paragraphs 1 to 10, wherein said human     is receiving or has received statin treatment or has reduced     responsiveness to statin treatment. -   12. The method of paragraph 10 or 11, wherein said antibody or     antibody fragment is administered to the human separately or     simultaneously with said statin treatment. -   13. The method of any one of paragraphs 7 to 9, wherein said     biological sample comprises serum, blood, faeces, tissue, a cell,     urine and/or saliva of said human. -   14. The method of any one of paragraphs 1 to 13, wherein said human     is indicated as heterozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation (eg, I474V or     E670G), optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 28, or said human     is indicated as homozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation I474V or E670G in     SEQ ID NO: 1. -   15. The method of any one of paragraphs 1 to 14, wherein said human     has been diagnosed with at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   16. The method of any one of paragraphs 1 to 15, wherein said     antibody or antibody fragment treats or reduces the risk in said     human of at least one condition selected from a lipid disorder,     hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   17. The method of any one of paragraphs 1 to 16, wherein the     nucleotide sequence s SEQ ID NO: 29 or 30. -   18. The method of any one of paragraphs 1 to 17, wherein said     antibody or antibody fragment is administered by intravenous or     subcutaneous administration and/or is comprised in an injectable     preparation.

Further exemplary ligands are in the paragraphs 1-17 as follows.

-   1. An antibody or antibody fragment for use in a method of (a)     reducing cholesterol level or maintaining previously reduced     cholesterol level in a human in need thereof or (b) targeting PCSK9     in a human,     -   wherein the antibody or fragment comprises a human kappa light         chain constant region that comprises a Val corresponding to         position 84 of SEQ ID NO: 50 or a Cys corresponding to position         87 of SEQ ID NO: 50, and the antibody or fragment specifically         binds a proprotein convertase subtilisin/kexin type 9 (PCSK9)         amino acid sequence that comprises a C-terminal domain         comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID         NO:1, wherein the human comprises a nucleotide sequence encoding         said amino acid sequence and comprises an IGKC*01 human light         chain constant region gene segment, or the human expresses         antibodies comprising human kappa light chain constant regions         comprising such an Val and Cys.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

An example provides:—

An antibody or antibody fragment for use in a method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

wherein the antibody or fragment comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V in SEQ ID NO:1, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGKC*01 human light chain constant region gene segment. Optionally, the antibody or fragment comprises an IGKC*01 human light chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

Another example provides:—

An antibody or antibody fragment for use in a method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

wherein the antibody or fragment comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation E670G in SEQ ID NO:1, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGKC*01 human light chain constant region gene segment. Optionally, the antibody or fragment comprises an IGKC*01 human light chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

In an example, said antibody or antibody fragment has been determined to specifically bind a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody or fragment comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50 and wherein said human comprises (i) an IGKC*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1.

-   2. The antibody or antibody fragment of paragraph 1, wherein the     antibody comprises a human kappa light chain constant region that     comprises a Val corresponding to position 84 of SEQ ID NO: 50 or a     Cys corresponding to position 87 of SEQ ID NO: 50. -   3. The antibody or antibody fragment of paragraph 1 or 2, wherein     the antibody comprises an IGKC*01 human kappa chain constant region. -   4. The antibody or antibody fragment of any one of paragraphs 1 to     3, wherein the human has been determined to comprise the nucleotide     sequence that encodes a PCSK9 comprising a C-terminal domain     comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1 and/or     a proprotein convertase subtilisin/kexin type 9 (PCSK9) variant     protein encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. -   5. The antibody or antibody fragment of any one of paragraphs 1 to     4, the method comprising the step of determining that the human     comprises the nucleotide sequence that encodes a PCSK9 comprising a     C-terminal domain comprising said mutation (eg, I474V or E670G)     and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)     variant protein comprising said mutation (eg, I474V or E670G),     optionally, wherein the determining step is performed before     administration of the antibody to the human. -   6. The antibody or antibody fragment of paragraph 5, wherein the     step of determining comprises assaying a biological sample from the     human for a nucleotide sequence encoding the PCSK9 that comprises     the C-terminal domain comprising the mutation (eg, I474V or E670G)     in SEQ ID NO: 1. -   7. The antibody or antibody fragment of paragraph 6, wherein the     assaying comprises contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   8. The antibody or antibody fragment of paragraph 6 or 7, wherein     the assaying comprises nucleic acid amplification and optionally one     or more methods selected from sequencing, next generation     sequencing, nucleic acid hybridization, and allele-specific     amplification and/or wherein the assaying is performed in a     multiplex format. -   9. The antibody or antibody fragment of any one of paragraphs 1 to     8, wherein said antibody or antibody fragment is for administration     to a human that is or has been further determined to be     substantially resistant to statin treatment. -   10. The antibody or antibody fragment of any one of paragraphs 1 to     9, wherein antibody or antibody fragment is for administration to a     human that is receiving or has received statin treatment or has     reduced responsiveness to statin treatment. -   11. The antibody or antibody fragment of paragraph 9 or 10, wherein     said antibody or antibody fragment is for administration to the     human separately or simultaneously with said statin treatment. -   12. The antibody or antibody fragment of any one of paragraphs 6 to     10, wherein said biological sample comprises serum, blood, faeces,     tissue, a cell, urine and/or saliva of said human. -   13. The antibody or antibody fragment of any one of paragraphs 1 to     12, wherein said human is indicated as heterozygous for a nucleotide     sequence encoding the PCSK9 C-terminal domain comprising a said     PCSK9 mutation (eg, I474V or E670G), optionally, wherein said human     is further indicated as comprising the nucleotide sequence of SEQ ID     NO: 28, or said human is indicated as homozygous for a nucleotide     sequence encoding the PCSK9 C-terminal domain comprising a mutation     I474V or E670G in SEQ ID NO: 1. -   14. The antibody or antibody fragment of any one of paragraphs 1 to     13, wherein said human has been diagnosed with at least one     condition selected from a lipid disorder, hyperlipoproteinemia,     hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,     a stroke, coronary heart disease, atherosclerosis, peripheral     vascular disease, claudication (eg, claudication associated with     elevated cholesterol) and high blood pressure. -   15. The antibody or antibody fragment of any one of paragraphs 1 to     14, wherein said antibody or antibody fragment treats or reduces the     risk in said human of at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   16. The antibody or antibody fragment of any one of paragraphs 1 to     15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30. -   17. The antibody or antibody fragment of any one of paragraphs 1 to     16, wherein said antibody or antibody fragment is for administration     by intravenous or subcutaneous administration and/or is comprised in     an injectable preparation.

Further exemplary methods are in the paragraphs 1-18 as follows.

-   1. A method of (a) reducing cholesterol level or maintaining     previously reduced cholesterol level in a human in need thereof     or (b) targeting PCSK9 in a human, the method comprising     administering to said human an antibody or antibody fragment that     specifically binds a proprotein convertase subtilisin/kexin type 9     (PCSK9) that comprises a C-terminal domain comprising a said PCSK9     mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody     or fragment comprises a human kappa light chain constant region that     comprises a Val corresponding to position 84 of SEQ ID NO: 50 or a     Cys corresponding to position 87 of SEQ ID NO: 50 and wherein said     human comprises (i) an IGKC*01 human light chain constant region     gene segment, or the human expresses antibodies comprising human     kappa light chain constant regions comprising such an Val and Cys     and (ii) a nucleotide sequence encoding said proprotein convertase     subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain     comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

An example provides:—

A method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation 1474 in SEQ ID NO: 1, wherein the antibody or fragment comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50 and wherein said human comprises (i) an IGKC*01 human light chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation I474V in SEQ ID NO: 1. Optionally, the antibody or fragment comprises an IGKC*01 human light chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

Another example provides:—

A method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation E670G in SEQ ID NO: 1, wherein the antibody or fragment comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50 and wherein said human comprises (i) an IGKC*01 human light chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation E670G in SEQ ID NO: 1. Optionally, the antibody or fragment comprises an IGKC*01 human light chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

In an example, said antibody or antibody fragment has been determined to specifically bind a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody or fragment comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50 and wherein said human comprises (i) an IGKC*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1.

-   2. The method of paragraph 1, comprising, before said administering,     selecting said human comprising said nucleotide sequence of (ii). -   3. The method of paragraph 1 or 2, wherein the antibody comprises a     human kappa light chain constant region that comprises a Val     corresponding to position 84 of SEQ ID NO: 50 or a Cys corresponding     to position 87 of SEQ ID NO: 50. -   4. The method of paragraph 1, 2 or 3, wherein the antibody comprises     an IGKC*01 human kappa chain constant region. -   5. The method of any one of paragraphs 1 to 4, wherein the human has     been determined to comprise the nucleotide sequence that encodes a     PCSK9 comprising a C-terminal domain comprising said mutation (eg,     I474V or E670G) in SEQ ID NO: 1 and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein encoded by the     nucleotide sequence of SEQ ID NO: 29 or 30. -   6. The method of any one of paragraphs 1 to 5, comprising the step     of determining that the human comprises the nucleotide sequence that     encodes a PCSK9 comprising a C-terminal domain comprising said     mutation (eg, I474V or E670G) and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein comprising said     mutation (eg, I474V or E670G), optionally, wherein the determining     step is performed before administration of the antibody to the     human. -   7. The method of paragraph 6, wherein the step of determining     comprises assaying a biological sample from the human for a     nucleotide sequence encoding the PCSK9 that comprises the C-terminal     domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1. -   8. The method of paragraph 7, wherein the assaying comprises     contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   9. The method of paragraph 7 or 8, wherein the assaying comprises     nucleic acid amplification and optionally one or more methods     selected from sequencing, next generation sequencing, nucleic acid     hybridization, and allele-specific amplification and/or wherein the     assaying is performed in a multiplex format. -   10. The method of any one of paragraphs 1 to 9, wherein said human     is or has been further determined to be substantially resistant to     statin treatment. -   11. The method of any one of paragraphs 1 to 10, wherein said human     is receiving or has received statin treatment or has reduced     responsiveness to statin treatment. -   12. The method of paragraph 10 or 11, wherein said antibody or     antibody fragment is administered to the human separately or     simultaneously with said statin treatment. -   13. The method of any one of paragraphs 7 to 9, wherein said     biological sample comprises serum, blood, faeces, tissue, a cell,     urine and/or saliva of said human. -   14. The method of any one of paragraphs 1 to 13, wherein said human     is indicated as heterozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation (eg, I474V or     E670G), optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 28, or said human     is indicated as homozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation I474V or -   E670G in SEQ ID NO: 1. -   15. The method of any one of paragraphs 1 to 14, wherein said human     has been diagnosed with at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   16. The method of any one of paragraphs 1 to 15, wherein said     antibody or antibody fragment treats or reduces the risk in said     human of at least one condition selected from a lipid disorder,     hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   17. The method of any one of paragraphs 1 to 16, wherein the     nucleotide sequence is SEQ ID NO: 29 or 30. -   18. The method of any one of paragraphs 1 to 17, wherein said     antibody or antibody fragment is administered by intravenous or     subcutaneous administration and/or is comprised in an injectable     preparation.

Further exemplary ligands are in the paragraphs 1-15 as follows.

-   1. An antibody or antibody fragment for use in a method of (a)     reducing cholesterol level or maintaining previously reduced     cholesterol level in a human in need thereof or (b) targeting PCSK9     in a human,     -   wherein the antibody or fragment comprises a human IGLC2*01         lambda light chain constant region, and the antibody or fragment         specifically binds a proprotein convertase subtilisin/kexin type         9 (PCSK9) amino acid sequence that comprises a C-terminal domain         comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID         NO:1, wherein the human comprises a nucleotide sequence encoding         said amino acid sequence and comprises a human IGLC2*01 lambda         light chain constant region gene segment, or the human expresses         antibodies comprising human IGLC2*01 lambda light chain constant         regions.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

An example provides:—

An antibody or antibody fragment for use in a method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

wherein the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V in SEQ ID NO:1, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises a human IGLC2*01 lambda light chain constant region gene segment. Optionally, the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

Another example provides:—

An antibody or antibody fragment for use in a method of (a) reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof or (b) targeting PCSK9 in a human,

wherein the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation E670G in SEQ ID NO:1, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises a human IGLC2*01 lambda light chain constant region gene segment. Optionally, the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

In an example, said antibody or antibody fragment has been determined to specifically bind a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region and wherein said human comprises (i) an IGLC2*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1.

-   2. The antibody or antibody fragment of paragraph 1, wherein the     human has been determined to comprise the nucleotide sequence that     encodes a PCSK9 comprising a C-terminal domain comprising said     mutation (eg, I474V or E670G) in SEQ ID NO: 1 and/or a proprotein     convertase subtilisin/kexin type 9 (PCSK9) variant protein encoded     by the nucleotide sequence of SEQ ID NO: 29 or 30. -   3. The antibody or antibody fragment of paragraphs 1 or 2, the     method comprising the step of determining that the human comprises     the nucleotide sequence that encodes a PCSK9 comprising a C-terminal     domain comprising said mutation (eg, I474V or E670G) and/or a     proprotein convertase subtilisin/kexin type 9 (PCSK9) variant     protein comprising said mutation (eg, I474V or E670G), optionally,     wherein the determining step is performed before administration of     the antibody to the human. -   4. The antibody or antibody fragment of paragraph 3, wherein the     step of determining comprises assaying a biological sample from the     human for a nucleotide sequence encoding the PCSK9 that comprises     the C-terminal domain comprising the mutation (eg, I474V or E670G)     in SEQ ID NO: 1. -   5. The antibody or antibody fragment of paragraph 4, wherein the     assaying comprises contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   6. The antibody or antibody fragment of paragraph 4 or 5, wherein     the assaying comprises nucleic acid amplification and optionally one     or more methods selected from sequencing, next generation     sequencing, nucleic acid hybridization, and allele-specific     amplification and/or wherein the assaying is performed in a     multiplex format. -   7. The antibody or antibody fragment of any one of paragraphs 1 to     6, wherein said antibody or antibody fragment is for administration     to a human that is or has been further determined to be     substantially resistant to statin treatment. -   8. The antibody or antibody fragment of any one of paragraphs 1 to     7, wherein antibody or antibody fragment is for administration to a     human that is receiving or has received statin treatment or has     reduced responsiveness to statin treatment. -   9. The antibody or antibody fragment of paragraph 7 or 8, wherein     said antibody or antibody fragment is for administration to the     human separately or simultaneously with said statin treatment. -   10. The antibody or antibody fragment of any one of paragraphs 4 to     8, wherein said biological sample comprises serum, blood, faeces,     tissue, a cell, urine and/or saliva of said human. -   11. The antibody or antibody fragment of any one of paragraphs 1 to     10, wherein said human is indicated as heterozygous for a nucleotide     sequence encoding the PCSK9 C-terminal domain comprising a said     PCSK9 mutation (eg, I474V or E670G), optionally, wherein said human     is further indicated as comprising the nucleotide sequence of SEQ ID     NO: 28, or said human is indicated as homozygous for a nucleotide     sequence encoding the PCSK9 C-terminal domain comprising a mutation     I474V or E670G in SEQ ID NO: 1. -   12. The antibody or antibody fragment of any one of paragraphs 1 to     11, wherein said human has been diagnosed with at least one     condition selected from a lipid disorder, hyperlipoproteinemia,     hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,     a stroke, coronary heart disease, atherosclerosis, peripheral     vascular disease, claudication (eg, claudication associated with     elevated cholesterol) and high blood pressure. -   13. The antibody or antibody fragment of any one of paragraphs 1 to     12, wherein said antibody or antibody fragment treats or reduces the     risk in said human of at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   14. The antibody or antibody fragment of any one of paragraphs 1 to     13, wherein the nucleotide sequence is SEQ ID NO: 29 or 30. -   15. The antibody or antibody fragment of any one of paragraphs 1 to     14, wherein said antibody or antibody fragment is for administration     by intravenous or subcutaneous administration and/or is comprised in     an injectable preparation.

Further exemplary methods are in the paragraphs 1-16 as follows.

-   1. A method of reducing cholesterol level or maintaining previously     reduced cholesterol level in a human in need thereof, the method     comprising administering to said human an antibody or antibody     fragment that specifically binds a proprotein convertase     subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain     comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO:     1, wherein the antibody or fragment comprises a human IGLC2*01     lambda light chain constant region and wherein said human     comprises (i) an IGLC2*01 human light chain constant region gene     segment, or the human expresses antibodies comprising a human     IGLC2*01 lambda light chain constant regions and (ii) a nucleotide     sequence encoding said proprotein convertase subtilisin/kexin type 9     (PCSK9) that comprises a C-terminal domain comprising said mutation     (eg, I474V or E670G) in SEQ ID NO: 1.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G.

An example provides:—

A method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation 1474 in SEQ ID NO: 1, wherein the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region and wherein said human comprises (i) an IGLC2*01 human light chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation I474V in SEQ ID NO: 1. Optionally, the antibody or fragment comprises an IGLC2*01 human light chain constant region.

Another example provides:—

A method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation E670G in SEQ ID NO: 1, wherein the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region and wherein said human comprises (i) an IGLC2*01 human light chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation E670G in SEQ ID NO: 1. Optionally, the antibody or fragment comprises an IGLC2*01 human light chain constant region.

In an example, said antibody or antibody fragment has been determined to specifically bind a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region and wherein said human comprises (i) an IGLC2*01 human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1

-   2. The method of paragraph 1, comprising, before said administering,     selecting said human comprising said nucleotide sequence of (ii). -   3. The method of paragraph 1 or 2, wherein the human has been     determined to comprise the nucleotide sequence that encodes a PCSK9     comprising a C-terminal domain comprising said mutation (eg, I474V     or E670G) in SEQ ID NO: 1 and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein encoded by the     nucleotide sequence of SEQ ID NO: 29 or 30. -   4. The method of any one of paragraphs 1 to 3, comprising the step     of determining that the human comprises the nucleotide sequence that     encodes a PCSK9 comprising a C-terminal domain comprising said     mutation (eg, I474V or E670G) and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein comprising said     mutation (eg, I474V or E670G), optionally, wherein the determining     step is performed before administration of the antibody to the     human. -   5. The method of paragraph 4, wherein the step of determining     comprises assaying a biological sample from the human for a     nucleotide sequence encoding the PCSK9 that comprises the C-terminal     domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1. -   6. The method of paragraph 5, wherein the assaying comprises     contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   7. The method of paragraph 5 or 6, wherein the assaying comprises     nucleic acid amplification and optionally one or more methods     selected from sequencing, next generation sequencing, nucleic acid     hybridization, and allele-specific amplification and/or wherein the     assaying is performed in a multiplex format. -   8. The method of any one of paragraphs 1 to 7, wherein said human is     or has been further determined to be substantially resistant to     statin treatment. -   9. The method of any one of paragraphs 1 to 8, wherein said human is     receiving or has received statin treatment or has reduced     responsiveness to statin treatment. -   10. The method of paragraph 8 or 9, wherein said antibody or     antibody fragment is administered to the human separately or     simultaneously with said statin treatment. -   11. The method of any one of paragraphs 5 to 7, wherein said     biological sample comprises serum, blood, faeces, tissue, a cell,     urine and/or saliva of said human. -   12. The method of any one of paragraphs 1 to 11, wherein said human     is indicated as heterozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation (eg, I474V or     E670G), optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 28, or said human     is indicated as homozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation I474V or E670G in     SEQ ID NO: 1. -   13. The method of any one of paragraphs 1 to 12, wherein said human     has been diagnosed with at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   14. The method of any one of paragraphs 1 to 13, wherein said     antibody or antibody fragment treats or reduces the risk in said     human of at least one condition selected from a lipid disorder,     hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   15. The method of any one of paragraphs 1 to 14, wherein the     nucleotide sequence is SEQ ID NO: 29 or 30. -   16. The method of any one of paragraphs 1 to 15, wherein said     antibody or antibody fragment is administered by intravenous or     subcutaneous administration and/or is comprised in an injectable     preparation.

Further exemplary ligands are in the paragraphs 1-15 as follows.

-   1. An antibody or antibody fragment for use in a method of (a)     reducing cholesterol level or maintaining previously reduced     cholesterol level in a human in need thereof or (b) targeting PCSK9     in a human, wherein the antibody or fragment comprises a human     variable domain that is derived from the recombination of a human VH     gene segment, a human D gene segment and a human JH gene segment,     wherein the VH gene segment is as defined in any one of clauses 80     to 90, and the antibody or fragment specifically binds a proprotein     convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that     comprises a C-terminal domain comprising a said PCSK9 mutation (eg,     I474V or E670G) in SEQ ID NO:1, wherein the human comprises a     nucleotide sequence encoding said amino acid sequence, and comprises     said VH gene segment or expresses antibodies comprising VH domains     that are derived from the recombination of said human VH gene     segment, a human D gene segment and a human JH gene segment.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

-   2. The antibody or antibody fragment of paragraph 1, wherein the     human has been determined to comprise the nucleotide sequence that     encodes a PCSK9 comprising a C-terminal domain comprising said     mutation (eg, I474V or E670G) in SEQ ID NO: 1 and/or a proprotein     convertase subtilisin/kexin type 9 (PCSK9) variant protein encoded     by the nucleotide sequence of SEQ ID NO: 29 or 30. -   3. The antibody or antibody fragment of paragraphs 1 or 2, the     method comprising the step of determining that the human comprises     the nucleotide sequence that encodes a PCSK9 comprising a C-terminal     domain comprising said mutation (eg, I474V or E670G) and/or a     proprotein convertase subtilisin/kexin type 9 (PCSK9) variant     protein comprising said mutation (eg, I474V or E670G), optionally,     wherein the determining step is performed before administration of     the antibody to the human. -   4. The antibody or antibody fragment of paragraph 3, wherein the     step of determining comprises assaying a biological sample from the     human for a nucleotide sequence encoding the PCSK9 that comprises     the C-terminal domain comprising the mutation (eg, I474V or E670G)     in SEQ ID NO: 1. -   5. The antibody or antibody fragment of paragraph 4, wherein the     assaying comprises contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and     -   detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1. -   6. The antibody or antibody fragment of paragraph 4 or 5, wherein     the assaying comprises nucleic acid amplification and optionally one     or more methods selected from sequencing, next generation     sequencing, nucleic acid hybridization, and allele-specific     amplification and/or wherein the assaying is performed in a     multiplex format. -   7. The antibody or antibody fragment of any one of paragraphs 1 to     6, wherein said antibody or antibody fragment is for administration     to a human that is or has been further determined to be     substantially resistant to statin treatment. -   8. The antibody or antibody fragment of any one of paragraphs 1 to     7, wherein antibody or antibody fragment is for administration to a     human that is receiving or has received statin treatment or has     reduced responsiveness to statin treatment. -   9. The antibody or antibody fragment of paragraph 7 or 8, wherein     said antibody or antibody fragment is for administration to the     human separately or simultaneously with said statin treatment. -   10. The antibody or antibody fragment of any one of paragraphs 4 to     8, wherein said biological sample comprises serum, blood, faeces,     tissue, a cell, urine and/or saliva of said human. -   11. The antibody or antibody fragment of any one of paragraphs 1 to     10, wherein said human is indicated as heterozygous for a nucleotide     sequence encoding the PCSK9 C-terminal domain comprising a said     PCSK9 mutation (eg, I474V or E670G), optionally, wherein said human     is further indicated as comprising the nucleotide sequence of SEQ ID     NO: 28, or said human is indicated as homozygous for a nucleotide     sequence encoding the PCSK9 C-terminal domain comprising a mutation     I474V or E670G in SEQ ID NO: 1. -   12. The antibody or antibody fragment of any one of paragraphs 1 to     11, wherein said human has been diagnosed with at least one     condition selected from a lipid disorder, hyperlipoproteinemia,     hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,     a stroke, coronary heart disease, atherosclerosis, peripheral     vascular disease, claudication (eg, claudication associated with     elevated cholesterol) and high blood pressure. -   13. The antibody or antibody fragment of any one of paragraphs 1 to     12, wherein said antibody or antibody fragment treats or reduces the     risk in said human of at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   14. The antibody or antibody fragment of any one of paragraphs 1 to     13, wherein the nucleotide sequence is SEQ ID NO: 29 or 30. -   15. The antibody or antibody fragment of any one of paragraphs 1 to     14, wherein said antibody or antibody fragment is for administration     by intravenous or subcutaneous administration and/or is comprised in     an injectable preparation.

Further exemplary methods are in the paragraphs 1-16 as follows.

-   1. A method of (a) reducing cholesterol level or maintaining     previously reduced cholesterol level in a human in need thereof     or (b) targeting PCSK9 in a human, the method comprising     administering to said human an antibody or antibody fragment that     specifically binds a proprotein convertase subtilisin/kexin type 9     (PCSK9) that comprises a C-terminal domain comprising a said PCSK9     mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody     or fragment comprises a human variable domain that is derived from     the recombination of a human VH gene segment, a human D gene segment     and a human JH gene segment, wherein the VH gene segment is as     defined in any one of clauses 80 to 90 and wherein said human     comprises (i) comprises said VH gene segment or expresses antibodies     comprising VH domains that are derived from the recombination of     said human VH gene segment, a human D gene segment and a human JH     gene segment and (ii) a nucleotide sequence encoding said proprotein     convertase subtilisin/kexin type 9 (PCSK9) that comprises a     C-terminal domain comprising said mutation (eg, I474V or E670G) in     SEQ ID NO: 1.

In an embodiment, said mutation is I474V. In another embodiment, said mutation is E670G.

In an embodiment of (b), the antibody or fragment treats or reduces cholesterol level or maintains previously reduced cholesterol level in the human.

-   2. The method of paragraph 1, comprising, before said administering,     selecting said human comprising said nucleotide sequence of (ii). -   3. The method of paragraph 1 or 2, wherein the human has been     determined to comprise the nucleotide sequence that encodes a PCSK9     comprising a C-terminal domain comprising said mutation (eg, I474V     or E670G) in SEQ ID NO: 1 and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein encoded by the     nucleotide sequence of SEQ ID NO: 29 or 30. -   4. The method of any one of paragraphs 1 to 3, comprising the step     of determining that the human comprises the nucleotide sequence that     encodes a PCSK9 comprising a C-terminal domain comprising said     mutation (eg, I474V or E670G) and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein comprising said     mutation (eg, I474V or E670G), optionally, wherein the determining     step is performed before administration of the antibody to the     human. -   5. The method of paragraph 4, wherein the step of determining     comprises assaying a biological sample from the human for a     nucleotide sequence encoding the PCSK9 that comprises the C-terminal     domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1. -   6. The method of paragraph 5, wherein the assaying comprises     contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   7. The method of paragraph 5 or 6, wherein the assaying comprises     nucleic acid amplification and optionally one or more methods     selected from sequencing, next generation sequencing, nucleic acid     hybridization, and allele-specific amplification and/or wherein the     assaying is performed in a multiplex format. -   8. The method of any one of paragraphs 1 to 7, wherein said human is     or has been further determined to be substantially resistant to     statin treatment. -   9. The method of any one of paragraphs 1 to 8, wherein said human is     receiving or has received statin treatment or has reduced     responsiveness to statin treatment. -   10. The method of paragraph 8 or 9, wherein said antibody or     antibody fragment is administered to the human separately or     simultaneously with said statin treatment. -   11. The method of any one of paragraphs 5 to 7, wherein said     biological sample comprises serum, blood, faeces, tissue, a cell,     urine and/or saliva of said human. -   12. The method of any one of paragraphs 1 to 11, wherein said human     is indicated as heterozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation (eg, I474V or     E670G), optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 28, or said human     is indicated as homozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation I474V or E670G in     SEQ ID NO: 1. -   13. The method of any one of paragraphs 1 to 12, wherein said human     has been diagnosed with at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   14. The method of any one of paragraphs 1 to 13, wherein said     antibody or antibody fragment treats or reduces the risk in said     human of at least one condition selected from a lipid disorder,     hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   15. The method of any one of paragraphs 1 to 14, wherein the     nucleotide sequence is SEQ ID NO: 29 or 30. -   16. The method of any one of paragraphs 1 to 15, wherein said     antibody or antibody fragment is administered by intravenous or     subcutaneous administration and/or is comprised in an injectable     preparation.

In an alternative, instead of being a method “of reducing cholesterol level or maintaining previously reduced cholesterol level “as rectied herein the method is “of treating and/or preventing a human PCSK9-mediated condition or disease” (eg, associated with hyper- or hypocholesterolemia or any other PCSK9-mediated disease or condition disclosed herein) and the mutation is instead selected from the group of R46L, A53V, N425S, A443T, I474V, Q619P and E670G. Hence in this alternative, any such embodiment is disclosed as written herein but with the mutation recited as selected from this group. In an example, the mutation is R46L. In an example, the mutation is A53V. In an example, the mutation is N425S. In an example, the mutation is A443T. In an example, the mutation is I474V. In an example, the mutation is Q619P. In an example, the mutation is E670G.

Regimens

A: The invention further provides the following regimens, ligands and kits.

-   1. A method for treating a human PCSK9-mediated disease or condition     in a human by targeting a rare variant human PCSK9, the method     comprising administering to the human a ligand (eg, an antibody or     fragment) that has been determined to specifically bind to said     PCSK9variant; wherein the human expresses said PCSK9 variant or the     genome of the human comprises a nucleotide sequence encoding said     PCSK9 variant; wherein said human is treated for said disease or     condition.

In an alternative, clause 1 provides:—

A method for targeting a rare variant human PCSK9, the method comprising administering to the human a ligand (eg, an antibody or fragment) that has been determined to specifically bind to said PCSK9variant; wherein the human expresses said PCSK9 variant or the genome of the human comprises a nucleotide sequence encoding said PCSK9 variant. In an embodiment, said human is treated for said disease or condition.

The variant PCSK9 can, for example, be any rare variant as described herein.

For example, there is provided:

1a. A method for treating a PCSK9-mediated disease or condition in a human by targeting a PCSK9 that comprises a C-terminal domain amino acid polymorphism (compared to SEQ ID NO: 1), the method comprising administering to the human a ligand (eg, an antibody or fragment) that has been determined to specifically bind to a PCSK9 comprising a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or 670G) (numbering according to SEQ ID NO:1); wherein the human expresses said PCSK9 or the genome of the human comprises a nucleotide sequence encoding said PCSK9; wherein said human is treated for said disease or condition.

For example, there is provided:

1b. A method for targeting a PCSK9 that comprises a C-terminal domain amino acid polymorphism (compared to SEQ ID NO: 1), the method comprising administering to the human a ligand (eg, an antibody or fragment) that has been determined to specifically bind to a PCSK9 comprising a C-terminal domain comprising a said PCSK9 mutation (eg, I474V or 670G) (numbering according to SEQ ID NO:1); wherein the human expresses said PCSK9 or the genome of the human comprises a nucleotide sequence encoding said PCSK9; optionally wherein said human is treated for said disease or condition.

In an embodiment, determination of said specific binding is by reference to binding assay data, eg, as determined using SPR or ELISA. Determination may, for example, be by reference to information in a printed publication, eg, with knowledge of data presented in the present or another patent application or in a journal article. Once armed with such knowledge (eg, in the absence of further testing of binding), the skilled person is able—by direction of the present invention—to treat a relevant human whose genotype or phenotype matches the binding specificity of the ligand.

The antibody or fragment can be according to any configuration, example, embodiment, aspect, clause or paragraph herein.

In an embodiment, the method comprises, before said administering, selecting a human comprising said nucleotide sequence encoding the PCSK9, wherein the human is said human in clause 1 (eg, la).

-   2. The method of clause 1, comprising before said administering the     step of determining that the ligand specifically binds to said     PCSK9, eg, using SPR or ELISA. -   3. The method of clause 1 or 2, wherein the specific binding to said     PCSK9 is binding with a dissociation constant (Kd) of 1 nM or less,     eg, 100, 10 or 1 pM or less. -   4. The method of any of clauses 1 to 3 (eg, clause 1a), wherein the     condition is elevated LDL-cholesterol or is caused by elevated     LDL-cholesterol. -   5. The method of clause 4 (eg, when dependent from clause 1a),     wherein the condition is selected from a lipid disorder,     hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   6. The method of any one of clauses 1 to 5 (eg, when dependent from     clause 1a), wherein the human has been determined to comprise the     nucleotide sequence that encodes a PCSK9 comprising a C-terminal     domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1     and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)     variant protein encoded by the nucleotide sequence of SEQ ID NO: 29     or 30. -   7. The method of any one of clauses 1 to 6 (eg, when dependent from     clause 1a), comprising the step of determining that the human     comprises the nucleotide sequence that encodes a PCSK9 comprising a     C-terminal domain comprising said mutation (eg, I474V or E670G)     and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)     variant protein comprising said mutation (eg, I474V or E670G),     optionally, wherein the determining step is performed before     administration of the antibody to the human. -   8. The method of clause 7, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding the PCSK9 that comprises the C-terminal domain     comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1. -   9. The method of clause 8, wherein the assaying comprises contacting     the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   10. The method of clause 8 or 9, wherein the assaying comprises     nucleic acid amplification and optionally one or more methods     selected from sequencing, next generation sequencing, nucleic acid     hybridization, and allele-specific amplification and/or wherein the     assaying is performed in a multiplex format. -   11. The method of any one of clauses 1 to 10, wherein said human is     or has been further determined to be substantially resistant to     statin treatment. -   12. The method of any one of clauses 1 to 11, wherein said human is     receiving or has received statin treatment or has reduced     responsiveness to statin treatment. -   13. The method of clauses 11 or 12, wherein said antibody or     antibody fragment is administered to the human separately or     simultaneously with said statin treatment. -   14. The method of any one of clauses 8 to 10, wherein said     biological sample comprises serum, blood, faeces, tissue, a cell,     urine and/or saliva of said human. -   15. The method of any one of clauses 1 to 14, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation (eg, I474V or     E670G), optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 28, or said human     is indicated as homozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation I474V or E670G in     SEQ ID NO: 1. -   16. The method of any one of clauses 1 to 15, wherein said human has     been diagnosed with at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   17. The method of any one of clauses 1 to 16, wherein said antibody     or antibody fragment treats or reduces the risk in said human of at     least one condition selected from a lipid disorder,     hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   18. The method of any one of clauses 1 to 17, wherein the nucleotide     sequence is SEQ ID NO: 29 or 30. -   19. The method of any one of clauses 1 to 18, wherein said antibody     or antibody fragment is administered by intravenous or subcutaneous     administration and/or is comprised in an injectable preparation. -   20. A ligand (eg, an antibody or fragment) for use in the method of     any one of clauses 1 to 19, wherein the ligand specifically binds     the PCSK9. -   21. A kit comprising the ligand of clause 20 and instructions for     carrying out the method of any one of clauses 1 to 19.

B: The invention further provides the following regimens, ligands and kits.

-   1. A method of reducing cholesterol level or maintaining a     previously reduced cholesterol level in a human in need thereof, the     method comprising:—     -   a. Carrying out an initial treatment of said human for an         initial treatment period by administering an anti-human PCSK9         ligand (eg, an antibody or fragment) to said human, wherein (i)         the ligand has been determined to specifically bind to a PCSK9         comprising a C-terminal domain comprising a said PCSK9 mutation         (eg, I474V or 670G) (numbering according to SEQ ID NO:1); (ii)         the human expresses said PCSK9 or the genome of the human         comprises a nucleotide sequence encoding said PCSK9 and (iii)         the human has received or is receiving statin treatment to lower         or maintain cholesterol level; wherein the initial treatment         comprises the administration of a single or multiple doses of         the ligand to the human;     -   b. Determining to (i) terminate statin treatment (ii) keep the         human off statin treatment; or (iii) reduce statin treatment         after said initial treatment period; and     -   c. Continuing to administer the ligand to said patient after         said time period has expired, thereby reducing cholesterol level         or maintaining a previously reduced cholesterol level in said         human.

In an embodiment, determination of said specific binding is by reference to binding assay data, eg, as determined using SPR or ELISA. Determination may, for example, be by reference to information in a printed publication, eg, with knowledge of data presented in the present or another patent application or in a journal article. Once armed with such knowledge (eg, in the absence of further testing of binding), the skilled person is able—by direction of the present invention—to treat a relevant human whose genotype or phenotype matches the binding specificity of the ligand.

The antibody or fragment can be according to any configuration, example, embodiment, aspect, clause or paragraph herein.

A pharmaceutically-effective amount of said ligand is administered.

In an embodiment, the method comprises, before said administering, selecting a human comprising said nucleotide sequence encoding the PCSK9, wherein the human is said human recited in clause 1.

In an example, the initial treatment period is 7 days, 14 days, 21 days, 28 days, a month, two months, three months, four months, five months, six months, seven months, eight months, nine months or a year.

-   2. The method of clause 1, wherein the human has or is suffering     from statin-associated memory loss or a statin-associated     neurodegenerative condition, or has or is at increased risk of     diabetes (eg, statin-associated diabetes). -   3. The method of clause 1 or 2, comprising, before said initial     treatment, the step of determining that the human has or is     suffering from statin-associated memory loss or a statin-associated     neurodegenerative condition, or has or is at increased risk of     diabetes (eg, statin-associated diabetes). -   4. The method of clause 2 or 3, comprising, after step (b) (eg,     during step (c)) determining that the memory loss or said     neurodegenerative condition has improved. -   5. The method of any one of clauses 1 to 4, wherein said human is     over 40 years of age (eg, 50 or over, 55 or over, 60 or over, 65 or     over, or 70 or over). -   6. The method of any one of clauses 1 to 5, wherein step (c)     comprises determining to increase the doses of said ligand to be     administered after said initial treatment period and administering     said increased doses to said human. -   7. The method of any one of clauses 1 to 6, wherein step (b)     comprises determining that the human is intolerant or refactory to     treatment by a statin. -   8. The method of any one of clauses 1 to 7, wherein the initial     treatment comprises the administration of a statin, fenofibrate (eg,     Tricor™ or Lofibra™) or ezetimibe to the human in addition to the     ligand. -   9. The method of any one of clauses 1 to 8, wherein step (b)     comprises terminating or reducing statin, fenofibrate (eg, Tricor™     or Lofibra™) or ezetimibe treatment during step (c). -   10. The method of any one of clauses 1 to 9, comprising increasing     (ie, increasing compared to the initial treatment dose) the ligand     dose during step (c). -   11. The method of any one of clauses 1 to 10, wherein the human has     received high dose statin treatment prior to the initial treatment,     and wherein step (c) comprises administering a lower (eg, a medium     or low) dose statin treatment in addition to said ligand.

The skilled person is familiar with the meaning of high, medium and low dose treatments (and how to determine according to each patient, eg, the patient's body mass). For example, the statin is selected from rosuvastatin, atorvastatin and simvastatin.

For example daily statin doses are as follows:—Low 10 to 20 mg (eg, 10 mg); medium >20 and <60 mg (eg, 40 mg); high 60-100 mg (eg, 80 mg).

-   12. The method of any one of clauses 1 to 10, wherein the human has     received medium dose statin treatment prior to the initial     treatment, and wherein step (c) comprises administering a lower (eg,     a low) dose statin treatment or no statin in addition to said     ligand. -   13. The method of any one of clauses 1 to 10, wherein the human has     received low dose statin treatment prior to the initial treatment,     and wherein step (c) comprises administering no statin in addition     to said ligand. -   14. The method of any one of clauses 1 to 13, comprising, before the     initial treatment, the step of determining that the ligand     specifically binds to said PCSK9, eg, using SPR or ELISA. -   15. The method of any one of clauses 1 to 14, wherein the specific     binding to said PCSK9 is binding with a dissociation constant (Kd)     of 1 nM or less, eg, 100, 10 or 1 pM or less. -   16. The method of any of clauses 1 to 15, wherein the human is at     risk of or suffering from a condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   17. The method of clause 16, wherein step (c) treats or reduces the     risk of said condition in the human. -   18. The method of any one of clauses 1 to 17, wherein the human has     been determined to comprise the nucleotide sequence that encodes a     PCSK9 comprising a C-terminal domain comprising said mutation (eg,     I474V or E670G) in SEQ ID NO: 1 and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein encoded by the     nucleotide sequence of SEQ ID NO: 29 or 30. -   19. The method of any one of clauses 1 to 18, comprising the step of     determining that the human comprises the nucleotide sequence that     encodes a PCSK9 comprising a C-terminal domain comprising said     mutation (eg, I474V or E670G) and/or a proprotein convertase     subtilisin/kexin type 9 (PCSK9) variant protein comprising said     mutation (eg, I474V or E670G), optionally, wherein the determining     step is performed before administration of the antibody to the     human. -   20. The method of clause 19, wherein the step of determining     comprises assaying a biological sample from the human for a     nucleotide sequence encoding the PCSK9 that comprises the C-terminal     domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1. -   21. The method of clause 20, wherein the assaying comprises     contacting the biological sample with     -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to at least one nucleotide         present in said selected sequence which is not present in SEQ ID         NO: 28 or hybridizes to an antisense sequence thereby forming a         complex when at least one nucleotide sequence encoding the PCSK9         that comprises the C-terminal domain comprising the mutation         (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         encoding the PCSK9 that comprises the C-terminal domain         comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises at         least one nucleotide present in said selected sequence which is         not present in SEQ ID NO: 28 thereby forming a complex when the         nucleotide sequence encoding the PCSK9 that comprises a         C-terminal domain comprising the mutation (eg, I474V or E670G)         in SEQ ID NO: 1 is present; and detecting the presence or         absence of the complex, wherein detecting the presence of the         complex determines that the human comprises the PCSK9 that         comprises the C-terminal domain comprising the mutation (eg,         I474V or E670G) in SEQ ID NO: 1. -   22. The method of clause 20 or 21, wherein the assaying comprises     nucleic acid amplification and optionally one or more methods     selected from sequencing, next generation sequencing, nucleic acid     hybridization, and allele-specific amplification and/or wherein the     assaying is performed in a multiplex format. -   23. The method of any one of clauses 1 to 22, wherein said human is     or has been further determined to be substantially resistant to     statin treatment. -   24. The method of any one of clauses 20 to 22, wherein said     biological sample comprises serum, blood, faeces, tissue, a cell,     urine and/or saliva of said human. -   25. The method of any one of clauses 1 to 24, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation (eg, I474V or     E670G), optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 28, or said human     is indicated as homozygous for a nucleotide sequence encoding the     PCSK9 C-terminal domain comprising said mutation I474V or E670G in     SEQ ID NO: 1. -   26. The method of any one of clauses 1 to 25, wherein said human has     been diagnosed with at least one condition selected from a lipid     disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,     hypercholesterolemia, a heart attack, a stroke, coronary heart     disease, atherosclerosis, peripheral vascular disease, claudication     (eg, claudication associated with elevated cholesterol) and high     blood pressure. -   27. The method of any one of clauses 1 to 26, wherein the nucleotide     sequence is SEQ ID NO: 29 or 30. -   28. The method of any one of clauses 1 to 27, wherein said ligand     (eg, antibody or antibody fragment) is administered by intravenous     or subcutaneous administration and/or is comprised in an injectable     preparation. -   29. A ligand (eg, an antibody or fragment) for use in the method of     any one of clauses 1 to 28, wherein the ligand specifically binds     the PCSK9. -   30. A kit comprising the ligand of clause 29 and instructions for     carrying out the method of any one of clauses 1 to 28.

In an example of any aspect of the invention, the ligand (eg, antibody or fragment, eg, alirocumab, bocovizumab or evolocumab, or an antibody or fragment comprising the variable domains of 316P or 31H4) is administered to the human at a two-weekly dose of from 75 to 150 mg (eg, from 75 to 150 mg administered once or twice over a two-week period). In an example, the ligand is for such administration to the human.

Determination of Specific Binding of Ligands of the Invention to PCSK9 Variants

Method of SPR Determination of Binding

Binding of the antibodies to the PCSK9 variants was carried out by SPR using the ProteOn XPR36™ Array system (BioRad). An anti-human IgG surface (Jackson Labs 109-005-008) was created on a GLC Biosensor chip by primary amine coupling. Test antibodies were captured on this surface as ligands. The PCSK9 variants were used as analytes and passed over the captured antibodies at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. Binding curves were double referenced using a buffer injection (i.e. 0 nM) to remove baseline drift and injection artefacts. Regeneration of the capture surface was with 100 mM phosphoric acid which removed the captured antibody allowing another cycle of capture and binding. The binding sensorgrams generated were analysed using the 1:1 model inherent to the ProteOn XPR36 Array system analysis software. The assay was performed at 25° C. and using 1×HBS-EP (Teknova) as running buffer.

Data

Three antibodies were tested and the resulting binding data are presented below (Table 3). Antibodies 316P and 31H4 are antibodies disclosed in US20110065902A1 (the sequences of these antibodies and their variable domains are incorporated herein by reference for possible use in the present invention and possible inclusion in claims herein). Antibody 316P comprises heavy chain variable domains derived from recombination of human VH3-23*04 and JH2*01 (with a D), and light chain variable domains derived from recombination of human Vκ4-1*01 and Jκ 2*01.

Evolocumab comprises a human IGHG2*01 heavy chain and a human IGLC2*01 lambda light chain, a VH derived from recombination of human IGHV1-18*01 and IGHJ6*01 (with a D segment) and a V derived from recombination of human IGLV2-14*01 and IGLJ2*01.

TABLE 3 SPR Determination of Ligand Binding Specificity for PCSK9 Variants Variant/Antibody ka (1/Ms) kd (1/s) KD (nM) PCSK9 a 316P 1.37E+06 2.75E−04 0.201 31H4 1.14E+06 6.38E−05 0.056 Evolocumab 1.14E+05 2.62E−05 0.229 PCSK9 a′ 316P 1.50E+06 2.72E−04 0.181 31H4 1.23E+06 6.06E−05 0.049 Evolocumab 1.24E+05 2.29E−05 0.185 PCSK9 c 316P 1.49E+06 2.75E−04 0.184 31H4 1.22E+06 5.69E−05 0.047 Evolocumab 1.20E+05 2.20E−05 0.183 PCSK9 r 316P 1.40E+06 2.76E−04 0.197 31H4 1.15E+06 5.82E−05 0.051 Evolocumab 1.16E+05 2.67E−05 0.230 PCSK9 f 316P 1.39E+06 2.82E−04 0.203 31H4 1.13E+06 5.95E−05 0.053 Evolocumab 1.16E+05 2.66E−05 0.229 PCSK9 p 316P 1.39E+06 2.73E−04 0.196 31H4 1.14E+06 6.12E−05 0.054 Evolocumab 1.14E+05 2.50E−05 0.219

Results

The results showed that all antibodies tested bound to PCSK9 variants equally, with any binding variation seen being within experimental error for such a strong affinity interaction.

Thus, the invention determines that an antibody with the following profile can specifically bind one or more variants of the invention:—1. An antibody (eg, 316P or alirocumab) that comprises heavy chain variable domains derived from recombination of human IGHV3-23*04 and IGHJH2*01 (with a D), and light chain variable domains derived from recombination of human IGKV4-1*01 and IGKJ2*01; or 2. An antibody (eg, evolocumab) that comprises human heavy chain variable domains derived from recombination of human IGHV1-18*01 and IGHJ6*01 (with a D segment) and light chain variable domains derived from recombination of human IGLV2-14*01 and IGLJ2*01.

Thus, according to the invention, the skilled person is hereby provided with the required determination of specific binding of the ligand to PCSK9 variants. Applications of this determination are set out above in the context of methods and other aspects of the invention.

REFERENCES

-   Horton et al, Trends Biochem Sci. 2007 February; 32(2):71-7. Epub     2007 Jan. 9, Molecular biology of PCSK9: its role in LDL metabolism. -   Seidah and Prat, J Mol Med (Berl). 2007 July; 85(7):685-96. Epub     2007 Mar. 10, The proprotein convertases are potential targets in     the treatment of dyslipidemia. -   Benjannet et al, J Biol Chem. 2004 Nov. 19; 279(47):48865-75. Epub     2004 Sep. 9, NARC-1/PCSK9 and its natural mutants: zymogen cleavage     and effects on the low density lipoprotein (LDL) receptor and LDL     cholesterol. -   Lagace et al, J Clin Invest. 2006 November; 116(11):2995-3005,     Secreted PCSK9 decreases the number of LDL receptors in hepatocytes     and in livers of parabiotic mice. -   Maxwell et al, Proc Natl Acad Sci USA. 2005 Feb. 8; 102(6):2069-74.     Epub 2005 Jan. 27, Overexpression of PCSK9 accelerates the     degradation of the LDLR in a post-endoplasmic reticulum compartment. -   Park et al, J Biol Chem. 2004 Nov. 26; 279(48):50630-8. Epub 2004     Sep. 22, Post-transcriptional regulation of low density lipoprotein     receptor protein by proprotein convertase subtilisin/kexin type 9a     in mouse liver. -   Rashid et al, Proc Natl Acad Sci USA. 2005 Apr. 12; 102(15):5374-9.     Epub 2005 Apr. 1, Decreased plasma cholesterol and hypersensitivity     to statins in mice lacking Pcsk9. -   Kotowski et al, Am J Hum Genet. 2006 March; 78(3):410-22. Epub 2006     Jan. 20, A spectrum of PCSK9 alleles contributes to plasma levels of     low-density lipoprotein cholesterol. -   Chen et al, J Am Coll Cardiol. 2005 May 17; 45(10):1611-9. Epub 2005     Apr. 21, A common PCSK9 haplotype, encompassing the E670G coding     single nucleotide polymorphism, is a novel genetic marker for plasma     low-density lipoprotein cholesterol levels and severity of coronary     atherosclerosis. -   Pisciotta et al, Atherosclerosis. 2006 June; 186(2):433-40. Epub     2005 Sep. 23, Additive effect of mutations in LDLR and PCSK9 genes     on the phenotype of familial hypercholesterolemia. -   Zhao et al, Am J Hum Genet. 2006 September; 79(3):514-23. Epub 2006     Jul. 18, Molecular characterization of loss-of-function mutations in     PCSK9 and identification of a compound heterozygote. -   Seidah et al, Proc Natl Acad Sci USA. 2003 Feb. 4; 100(3):928-33.     Epub 2003 Jan. 27, The secretory proprotein convertase neural     apoptosis-regulated convertase 1 (NARC-1): liver regeneration and     neuronal differentiation.

Example 2: IL4 Receptor Alpha (IL4Ra; CD124)

Interleukin-4 (IL-4, also known as B cell stimulating factor or BSF-1) was originally characterized by its ability to stimulate the proliferation of B cells in response to low concentrations of antibodies directed to surface immunoglobulin. IL-4 has been shown to possess a broad spectrum of biological activities, including growth stimulation of T cells, mast cells, granulocytes, megakaryocytes and erythrocytes. IL-4 induces the expression of class Il major histocompatibility complex molecules in resting B cells, and enhances the secretion of IgE and IgG1 isotypes by stimulated B cells

In an example, the invention provides a method of treating or reducing the risk of an IL4Ra-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human IL4RA protein. The invention also provides a corresponding ligand.

The present invention provides anti-IL4Ra ligands; and IL4Ra-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of IL4Ra, in particular human IL4Ra or its ligands and in screening assays to identify other antagonists of IL4Ra activity. Some of the ligands of the invention are useful for inhibiting binding of IL4Ra to IL4, or inhibiting IL4Ra-mediated activities.

Anti-IL4Ra ligands (eg, antibodies and anti-sense RNA) have been developed based on targeting and neutralising so-called “wild-type” human IL4Ra, which is a commonly-occurring form (see, eg, SEQ ID NO: 67). While such therapies are useful for human patients harbouring this form of human IL4RA, the inventor considered it useful to investigate the possibility of targeting rarer—but still naturally-occurring—forms of IL4Ra amongst human populations. In this way, the inventor arrived at insight into the natural occurrences and distributions of rarer human IL4Ra forms that can serve as useful targets (at the protein or nucleic acid level) for human treatment, prophylaxis and diagnosis pertinent to diseases and conditions mediated or associated with IL4RA activity. This particularly provides for tailored therapies, prophylaxis and diagnosis in humans that are devoid of the common IL4Ra gene or protein.

The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in activity and/or conformation of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to more effectively tailor medicines and diagnosis of patients. The invention, therefore, provides for tailored pharmaceuticals and testing that specifically addresses rarer IL4Ra polymorphic variant forms. Such forms or “alleles” (at the nucleotide level), comprise one or more changes at the nucleotide and amino acid levels from the corresponding common form nucleotide and amino acids sequences, ie, there are one or more non-synonymous changes at the nucleotide level that translate into one or more corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting such rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention.

With this realisation, the inventor realised that there is significant industrial and medical application for the invention in terms of guiding the choice of anti-IL4Ra ligand for administration to human patients for therapy and/or prophylaxis of IL4Ra-mediated or associated diseases or conditions. In this way, the patient receives drugs and ligands that are tailored to their needs—as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example the present inventor decided to determine a set of human IL4Ra variants on the basis of the following criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population. The inventor selected variants having at least 3 of the 4 following criteria:—

Naturally-occurring human IL4Ra variation having a cumulative human allele frequency of 35% or less;

Naturally-occurring human IL4Ra variation having a total human genotype frequency of about 50% or less;

Naturally-occurring human IL4Ra variation found in many different human ethnic populations (using the standard categorisation of the 1000 Genomes Project; see Table 2 below); and

Naturally-occurring human IL4Ra variation found in many individuals distributed across such many different ethnic populations.

On the basis of these criteria, the inventor identified variants listed in Table 11 below. The inventor's selection included, as an additional or alternative consideration, selection for nucleotide variation that produced amino acid variation in corresponding IL4Ra forms (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.

In an example, the invention provides a method of treating or reducing the risk of an IL4Ra-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. As explained further below, these amino acid variations are found in naturally-occurring IL-4Ra variants in humans found in many populations. Said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

An example also provides a ligand (eg, an antibody or antibody fragment) for treating or reducing the risk of an IL4Ra-mediated disease or condition in a human in need thereof, the method comprising administering to said human said ligand, wherein the ligand specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In an example, the invention provides a method of targeting IL4Ra in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In an example, the human is suffering from or at risk of an IL4Ra-mediated disease or condition. In an example, the method treats or reduces the risk of an IL4Ra-mediated disease or condition in the human.

An example also provides a ligand (eg, an antibody or antibody fragment) for targeting IL4Ra in a human, the method comprising administering to said human said ligand, wherein the ligand specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In an example, the human is suffering from or at risk of an IL4Ra-mediated disease or condition. In an example, the method treats or reduces the risk of an IL4Ra-mediated disease or condition in the human.

In an embodiment, (i) the antibody or fragment comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and wherein (ii) said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

Additionally or alternatively, in an embodiment, (i) the antibody or fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

The biological activities of IL-4 are mediated by specific cell surface receptors for IL-4. Human IL-4 receptor alpha (hIL-4Ra) is described in, for example, U.S. Pat. Nos. 5,599,905, 5,767,065, and 5,840,869. Antibodies to hIL-4R are described in U.S. Pat. No. 5,717,072.

Methods for using antibodies to hIL-4R are described in U.S. Pat. Nos. 5,714,146; 5,985,280; and 6,716,587.

The human IL-4Ra (aka IL-4Ra) subunit (Swiss Prot accession number P24394) is a 140 kDa type 1 membrane protein that binds human IL-4 with a high affinity (Andrews et al J. Biol. Chem (2002) 277:46073-46078). The IL-4/IL-4Ra complex can dimerize with either the common gamma chain (γc, CD132) or the IL-13Ralpha1 (IL-13Rα1) subunit, via domains on IL-4, to create two different signalling complexes, commonly referred to as Type I and Type II receptors, respectively. Alternatively, IL-13 can bind IL-13Rα1 to form an IL-13/IL-13Rα1 complex that recruits the IL-4Rα subunit to form a Type II receptor complex. Thus, IL-4Ra mediates the biological activities of both IL-4 and IL-13 (reviewed by Gessner et al, Immunobiology, 201:285, 2000). In vitro studies have shown that IL-4 and IL-13 activate effector functions in a number of cell types, for example in T cells, B cells, eosinophils, mast cells, basophils, airway smooth muscle cells, respiratory epithelial cells, lung fibroblasts, and endothelial cells (reviewed by Steinke et al, Resp Res, 2:66, 2001, and by Willis-Karp, Immunol Rev, 202:175, 2004).

IL-4 by binding to its receptor (IL-4R) is essential for the development of airway inflammation present in asthma, through the induction of IgE synthesis in B cells and differentiation of T cells to a Th2 phenotype. Dupilumab is a monoclonal antibody designed for the treatment of atopic diseases. It binds to the alpha subunit of the interleukin-4 receptor. Through blockade of IL-4R alpha, dupilumab modulates signaling of both the IL-4 and IL-13 pathway, which have been implicated in the pathophysiology of allergic disease. Anti-IL4Ra ligands, antibodies and fragments according to the invention can, for example, be used for treating or reducing the risk of allergic disease.

In an example, the ligand, antibody or fragment is for treating or reducing the risk of (or treats or reduces the risk of) allergic asthma, eosinophilic asthma or atopic dermatitis, optionally wherein the antibody is dupilumab.

In addition to its role in asthma, IL-4Ra has been linked with a number of other pathologies, e.g. as follows (anti-IL4Ra ligands, antibodies and fragments according to the invention can, for example, be used for treating or reducing the risk of any one of these diseases or conditions):

Chronic Obstructive Pulmonary Disease (COPD) includes patient populations with varying degrees of chronic bronchitis, small airway disease and emphysema and is characterised by progressive irreversible lung function decline that responds poorly to current asthma based therapy. The underlying causes of COPD remain poorly understood. The “Dutch hypothesis” proposes that there is a common susceptibility to COPD and asthma and therefore, that similar mechanisms may contribute to the pathogenesis of both disorders (Sluiter et al., Eur Respir J,). Zheng et al (J Clin Invest, 106(9):1081-93, 2000) have demonstrated that overexpression of IL-13 in the mouse lung caused emphysema, elevated mucus production and inflammation, reflecting aspects of human COPD. Furthermore, AHR, an IL-13 dependent response in murine models of allergic inflammation, has been shown to be predictive of lung function decline in smokers (Tashkin et al., Am J Respir Crit Care Med, 153(6 Pt 1):1802-11, 1996). A link has also been established between an IL-13 promoter polymorphism and susceptibility to develop COPD (Van Der Pouw Kraan et al., Genes Immun, 3(7): 436-9, 2002). The signs are therefore that IL-4/IL-13 pathway, and in particular IL-13, plays an important role in the pathogenesis of COPD.

In addition to asthma, the IL-4/Il-13 pathway has been linked to other fibrotic conditions, like systemic sclerosis (Hasegawa et al., J Rheumatol, 24(2):328-32, 1997), pulmonary fibrosis (Hancock et al., Am J Respir Cell Mol Biol, 18(1): 60-5, 1998), parasite-induced liver fibrosis (Fallon et al., J Immunol, 164(5): 2585-91, 2000; Chiaramonte et al., J Clin Invest, 104(6): 777-85, 1999; Chiaramonte Hepatology 34(2):273-82, 2001), and cystic fibrosis (Hauber et al. J. Cyst Fibr, 2:189, 2003).

IL-4 and to some extent IL-13, are crucial for B cell mediated activities, such as B cell proliferation, immunoglobulin secretion, and expression of FcepsilonR. Clinical applications of an IL-4Ra inhibitor include for example, use in allergy therapy to suppress IgE synthesis (including for example atopic dermatitis and food allergy), use in transplation therapy to prevent transplant rejection, as well as suppression of delayed-type hypersensitivity or contact hypersensitivity reactions.

Il-4R antagonists may also find use as adjuvants to allergy immunotherapy and as vaccine adjuvants.

IL-4Ra polymorphisms in the human population have been described (reviewed by Gessner et al, Immunobiology, 201:285, 2000) and association with IgE levels or clinical atopy has been reported in some populations. For instance, V75R576 IL-4Rα is associated with allergic asthma and enhanced IL-4Rα function (Risma et al. J. Immunol. 169(3):1604-1610, 2002).

Anti-IL4Ra ligands, antibodies and fragments according to the invention optionally neutralise IL-4Ra with high potency, for example as described in more detail in the Examples of EP2604628. Neutralisation means inhibition of a biological activity mediated by IL-4Ra. Ligands, antibodies and fragments according to the invention may neutralise one or more activities mediated by IL-4Ra. The inhibited biological activity is likely mediated by prevention of IL-4Ra forming a signalling complex with gamma chain (or IL-13Ra) and either of the associated soluble ligands, e.g. IL-4 or IL-13.

Neutralisation of IL-4 or IL-13 signalling through its IL-4Ra containing receptor complex may be measured by inhibition of IL-4 or IL-13 stimulated TF-1 cell proliferation.

The ligand, antibody or fragment according to the invention is, for example, dupilumab or any one disclosed in WO 08/054606 (Regeneron), EP2604628 (Medimmune), WO 01/92340 (immnunex) or WO 05/047331 (Immunex), the disclosures of which (including the sequences, eg, VH, VL and heavy and light chain sequences) are incorporated herein by reference for potential inclusion in one or more claims herein. According to a further aspect of the ligand, antibody or fragment according to the invention is capable of binding human interleukin-4 receptor alpha (hIL-4Ra) and cynomolgus monkey interleukin-4 receptor alpha (cyIL-4Ra). Cynomolgus IL-4Ra cDNA sequence is shown as SEQ ID NO: 455 EP2604628. In a particular embodiment the antibody or fragment is a human antibody or fragment. The ligand, antibody or fragment according to the invention is an antibody or fragment, for example, that comprises a VH and/or VL of dupilumab, or comprises a heavy and/or light chain of dupilumab. In one embodiment, the ligand, antibody or fragment comprises a VH domain comprising SEQ ID NO: 69. In one embodiment, the ligand, antibody or fragment comprises a VL domain comprising SEQ ID NO: 70. In one embodiment, the ligand, antibody or fragment comprises a heavy chain comprising SEQ ID NO: 71. In one embodiment, the ligand, antibody or fragment comprises a light chain comprising SEQ ID NO: 72.

In a specific embodiment, the ligand, antibody or fragment of present invention comprises an Fc region, wherein the Fc region comprises at least one non-native amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may comprise additional and/or alternative non-native amino acid residues known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

The ligand, antibody or fragment according to the invention is for treating or preventing or reducing the risk of (or treats or prevents or reduces the risk of), for example, any disease or condition disclosed in any of WO 08/054606 (Regeneron), EP2604628 (Medimmune), WO 01/92340 (immnunex) and WO 05/047331 (Immunex), the disclosures of which diseases and conditions are incorporated herein by reference for potential inclusion in one or more claims herein.

Further encompassed by the invention is the use of the ligand, antibody or fragment in the manufacture of a medicament for use to attenuate or inhibit an IL-4Ra-mediated disease or disorder in a human. IL-4Ra-mediated or related disorders which are treated by the ligand, antibody or fragment of the invention include, for example, arthritis (including septic arthritis), herpetiformis, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's Disease, benign prostate hyperplasia, lung disorders, such as mild, moderate or severe asthma, inflammatory disorders such as inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Grave's disease, pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephrosis.

Particular conditions for which a ligand, antibody or fragment of the invention may be used in treatment or diagnosis include: asthma, COPD (eg, chronic bronchitis, small airway disease or emphysema), inflammatory bowel disease, a fibrotic condition (eg, systemic sclerosis, pulmonary fibrosis, parasite-induced liver fibrosis, or cystic fibrosis), allergy (for example atopic dermatitis, dust mite allergy, pet allergy or food allergy), transplation therapy to prevent transplant rejection, suppression of a delayed-type hypersensitivity or a contact hypersensitivity reaction, as an adjuvant to allergy immunotherapy or as a vaccine adjuvant. In an example, the method and ligand are for treating or preventing (or reducing the risk of) nasaly polyps and/or sinusitis.

Thus, a ligand, antibody or fragment of the invention is useful as a therapeutic agent in the treatment of a condition involving IL-4, IL-13 or IL-4Ra expression and/or activity. One embodiment, among others, is a method of treatment comprising administering an effective amount of a ligand, antibody or fragment of the invention to a patient in need thereof, wherein functional consequences of IL-4Ra activation are decreased. Another embodiment, among others, is a method of treatment comprising (i) identifying a patient demonstrating IL-4, IL-13 or IL-4Ra expression or activity, and (ii) administering an effective amount of a ligand, antibody or fragment of the invention to the patient, wherein a functional consequence of IL-4Ra activation are attenuated. An effective amount according to the invention is an amount that modulates (e.g. decreases) the functional consequences of IL-4Ra activation so as to modulate (e.g. decrease or lessen) the severity of at least one symptom of the particular disease or disorder being treated, but not necessarily cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, comprising administering to a patient in need thereof an effective amount of one or more ligands, antibodies or fragments of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein such that the severity of at least one symptom of any of the disorders is reduced. Another embodiment of the invention, among others, is a method of antagonizing at least one effect of IL-4Ra comprising contacting with or administering an effective amount of one or more ligands, antibodies or fragments of the present invention such that said at least one effect of IL-4Ra is antagonized, e.g. the ability of IL-4Ra to form a complex (the precursor to active signalling) with IL-4.

A ligand, antibody or fragment of the invention can, in an example, be used in combination with another therapeutic agent for the treatment of cancer. Suitable agents to be used in combination include those disclosed in EP2604628 (eg, in paragaraph [0287]), which disclosure is incorporated herein by reference.

Tailoring Antibodies to Rarer IL4Ra Variant Profile

As outlined herein (for example, in the context of PCSK9 in Example 1), the invention includes the possibility to tailor treatment of humans further by selecting antibody-based ligands with variable domains and/or constant domains based on gene segments found in many humans of the ethnic populations where the variant TOI forms are found to meet the selection criteria of the invention. This also applies mutatis mutandis where the TOI is human IL4Ra, as in the present example. Thus, all disclosure herein relating to tailoring variable and/or constant domains apply to the present example, relating to IL4Ra and is combinable for use in one or more claims herein.

As described in Example 1, an example is provided for ligands comprising antibody VH domains derived from recombination of human IGHV gene segments comprising selected nucleotides at positions in the HCDR1 or FW3 where there is variability in humans (ie, where SNPs occur in humans).

Further information is provided in Table 4, which shows variation at these positions, as well as the variant distributions across the 1000 Genomes Project database relating to many human populations.

In other embodiments, as explained more fully above, the invention provides for ligands which are tailored to the human recipient's genotype and/or phenotype based on alternative human VH gene segments, or on Vκ, Vλ or constant region gene segments (see further Table 9 for representative variants).

In an example, following this guidance, the chosen ligand is dupilumab.

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23*04), a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40. (ii) wherein the ligand comprises a VH domain derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment, and wherein said human comprises a IGHV3-7*01 VH gene segment or the human expresses VH domains derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment. (iii) wherein the ligand comprises a Vκ domain derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment, and wherein said human comprises a IGKV1-12*01 Vκ gene segment or the human expresses Vκ domains derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment. (iv) wherein the ligand comprises a Vκ domain derived from the recombination of a human Vκ segment and a human Jκ segment, the human Vκ segment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36 and wherein said human comprises a Vκ gene segment encoding a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the human expresses Vκ domains that comprise a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 and wherein said human comprises a Vκ gene segment encoding a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 or the human expresses Vκ domains that comprise a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38. (v) wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4. (vi) wherein the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6. (vii) wherein the ligand comprises a human kappa chain constant region that comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16 and wherein said human comprises (i) an IGKC1*01 human kappa chain constant region gene segment, or the human expresses antibodies comprising human kappa chain constant regions comprising a Val corresponding to position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16. (viii) wherein the ligand comprises a human IGLC1*01 lambda chain constant region and wherein said human comprises (i) a human IGLC1*01 lambda chain constant region gene segment, or the human expresses antibodies comprising human IGLC1*01 lambda chain constant regions.

-   (ix) wherein the ligand comprises a human gamma-4 heavy chain     constant region that comprises a Leu at position 189 shown in SEQ ID     NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein     said human comprises (i) an IGHG4*01 human heavy chain constant     region gene segment, or the human expresses antibodies comprising     human gamma-4 heavy chain constant regions comprising a Leu at     position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown     in SEQ ID NO: 73.

For example, as per example (ix), the inventor identified the possibility of addressing IGH-gamma-4 variation and identified utility for variations 189L and 289R individually or in combination, since these residues are part of the CH3 domain, and as such they form part of antibody Fc regions. Thus, matching of these CH3 variations with the patient is especially beneficial for reasons as discussed above. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human gamma-4 heavy chain constant region that comprises a Leu corresponding to position 189 of SEQ ID NO: 73 or an Arg corresponding to position 289 of SEQ ID NO: 73 and wherein the genome of the human comprises a gamma-4 heavy chain constant region nucleotide sequence that encodes such a Leu and/or Arg or the human expresses antibodies comprising human gamma-4 constant regions comprising such a Leu and/or Arg. An example of such a ligand is dupilumab.

Determination of Specific Binding of Ligands of the Invention to IL4RA Variants

The specific binding of Ligands of the invention to IL4Ra variants can be performed using the SPR method described in Example 1.

Embodiments are provided as follows:—

Methods with VH Tailoring

-   1. A method of treating or reducing the risk of an IL4Ra-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a human IL4Ra protein     that comprises a mutation selected from the group consisting of     I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;     -   wherein (i) the ligand comprises a VH domain derived from the         recombination of a human VH segment, a human D gene segment and         a human JH segment, the human VH segment encoding the framework         1 of SEQ ID NO: 40 and wherein said human comprises a VH gene         segment encoding the framework 1 of SEQ ID NO: 40, or the human         expresses VH domains that comprise the framework 1 of SEQ ID NO:         40; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said IL4RA protein comprising said mutation selected from the         group consisting of I75V, E400A, C431R, S503P, Q576R and S752A         in SEQ ID NO: 67.

In an alternative, clause 1 provides:—

A method of targeting IL4Ra in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human IL4Ra protein that comprises a mutation selected from the group consisting of 175V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;

wherein (i) the ligand comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and

wherein (ii) said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of 175V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In an example, the human is suffering from or at risk of an IL4Ra-mediated disease or condition. In an example, the method treats or reduces the risk of an IL4Ra-mediated disease or condition in the human. Optionally the disease is asthma. Optionally the disease is atopic dermatitis. Optionally the disease is nasal polyps and/or sinusitis.

In an example of the method or ligand of the invention, the IL4Ra comprises mutation I75V. In an example of the method or ligand of the invention, the IL4Ra comprises mutation E400A. In an example of the method or ligand of the invention, the IL4Ra comprises mutation C431R. In an example of the method or ligand of the invention, the IL4Ra comprises mutation S503P. In an example of the method or ligand of the invention, the IL4Ra comprises mutation Q576R. In an example of the method or ligand of the invention, the IL4Ra comprises mutation S752A.

-   2. The method of clause 1, wherein the IL4Ra comprises mutations     I75V and Q576R in SEQ ID NO: 67 and optionally the disease is     asthma. Optionally the disease is atopic dermatitis. Optionally the     disease is nasal polyps and/or sinusitis.

Q576R and 175V are associated with airway inflammation disease, eg, asthma. For example, see Clin Exp Allergy. 2003 August; 33(8):1111-7, “Polymorphisms in the interleukin-4 and interleukin-4 receptor alpha chain genes confer susceptibility to asthma and atopy in a Caucasian population”, Begh6 B et al (referring to 175V instead as 150V); J Asthma. 2010 April; 47(3):238-44. doi: 10.3109/02770900903509099, “Association and gene-gene interactions of eight common single-nucleotide polymorphisms with pediatric asthma in middle china”, Wu X et al; Clin Exp Allergy. 2004 October; 34(10):1570-5, “Haplotypes of the interleukin-4 receptor alpha chain gene associate with susceptibility to and severity of atopic asthma”, Hytonen A M et al, all incorporated herein by reference.

-   3. The method of clause 1 or 2, wherein the nucleotide sequence     of (ii) comprises nucleotide mutation −3223T (dB SNP numbering).

This variation is associated with airway inflammation disease, eg, asthma.

-   4. The method of any preceding clause, wherein each said human VH     gene segment comprises SEQ ID NO: 39. -   5. The method of any preceding clause, wherein the VH gene segment     comprised by said human is a germline VH gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes an IL4Ra     protein comprising said mutation selected from the group consisting     of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67     and/or an IL4Ra protein comprising said mutation selected from the     group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in     SEQ ID NO: 67. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes an IL4Ra protein comprising said mutation selected from     the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A     in SEQ ID NO: 67 and/or (b) an IL4Ra protein comprising said     mutation said mutation selected from the group consisting of I75V,     E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, optionally,     wherein the determining step is performed before administration of     the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a IL4Ra protein comprising said mutation selected     from the group consisting of I75V, E400A, C431R, S503P, Q576R and     S752A in SEQ ID NO: 67. -   10. The method of clause 9, wherein the assaying comprises     contacting the biological sample with at least one oligonucleotide     probe comprising a sequence of at least 10 contiguous nucleotides of     a nucleotide sequence encoding an IL4Ra protein comprising said     mutation selected from the group consisting of I75V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67 or comprising an antisense     sequence of said contiguous nucleotides, wherein said sequence of     contiguous nucleotides comprises a nucleotide sequence encoding said     mutation selected from the group consisting of I75V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67, thereby forming a complex     when at least one nucleotide sequence encoding the IL4Ra protein     comprising said mutation selected from the group consisting of 175V,     E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 is present;     and detecting the presence or absence of the complex, wherein     detecting the presence of the complex determines that the human     comprises the IL4Ra protein comprising said mutation selected from     the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A     in SEQ ID NO: 67. -   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     IL4Ra protein comprising said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67, optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 68, or said human     is indicated as homozygous for a nucleotide sequence encoding the     IL4Ra protein comprising said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to an asthma     treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received an asthma treatment or has reduced     responsiveness to an asthma treatment. -   16. The method of any preceding clause, wherein said disease or     condition is an inflammatory disease or condition; an atopic disease     or condition; a respiratory disease or condition; a disease or     condition associated with elevated IgE; or a disease or condition     associated with elevated IL-4 and/or IL-13 activity. -   17. The method of any preceding clause, wherein said disease or     condition is selected from the group consisting of an airway     inflammatory disease or condition, chronic obstructive pulmonary     disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary     infiltrate with eosinophilia, environmental lung disease, pneumonia,     bronchiectasis, cystic fibrosis, interstitial lung disease, primary     pulmonary hypertension, pulmonary thromboembolism, disorders of the     pleura, disorders of the mediastinum, disorders of the diaphragm,     hypoventilation, hyperventilation, sleep apnea, acute respiratory     distress syndrome, mesothelioma, sarcoma, graft rejection, graft     versus host disease, lung cancer, allergic rhinitis, allergy,     asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic     bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary     fibrosis, invasive pneumococcal disease, influenza, nontuberculous     mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,     pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,     pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary     inflammation, pulmonary histiocytosis X, pulmonary hypertension,     pulmonary nocardiosis, pulmonary tuberculosis, pulmonary     veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and     Wegener's granulomatosis. -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition selected from the group     consisting of an airway inflammatory disease or condition, chronic     obstructive pulmonary disease, asthma, pneumonia, hypersensitivity     pneumonitis, pulmonary infiltrate with eosinophilia, environmental     lung disease, pneumonia, bronchiectasis, cystic fibrosis,     interstitial lung disease, primary pulmonary hypertension, pulmonary     thromboembolism, disorders of the pleura, disorders of the     mediastinum, disorders of the diaphragm, hypoventilation,     hyperventilation, sleep apnea, acute respiratory distress syndrome,     mesothelioma, sarcoma, graft rejection, graft versus host disease,     lung cancer, allergic rhinitis, allergy, asbestosis, aspergilloma,     aspergillosis, bronchiectasis, chronic bronchitis, emphysema,     eosinophilic pneumonia, idiopathic pulmonary fibrosis, invasive     pneumococcal disease, influenza, nontuberculous mycobacteria,     pleural effusion, pneumoconiosis, pneumocytosis, pneumonia,     pulmonary actinomycosis, pulmonary alveolar proteinosis, pulmonary     anthrax, pulmonary edema, pulmonary embolus, pulmonary inflammation,     pulmonary histiocytosis X, pulmonary hypertension, pulmonary     nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive     disease, rheumatoid lung disease, sarcoidosis, and Wegener's     granulomatosis. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of at     least one condition selected from the group consisting of an airway     inflammatory disease or condition, chronic obstructive pulmonary     disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary     infiltrate with eosinophilia, environmental lung disease, pneumonia,     bronchiectasis, cystic fibrosis, interstitial lung disease, primary     pulmonary hypertension, pulmonary thromboembolism, disorders of the     pleura, disorders of the mediastinum, disorders of the diaphragm,     hypoventilation, hyperventilation, sleep apnea, acute respiratory     distress syndrome, mesothelioma, sarcoma, graft rejection, graft     versus host disease, lung cancer, allergic rhinitis, allergy,     asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic     bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary     fibrosis, invasive pneumococcal disease, influenza, nontuberculous     mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,     pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,     pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary     inflammation, pulmonary histiocytosis X, pulmonary hypertension,     pulmonary nocardiosis, pulmonary tuberculosis, pulmonary     veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and     Wegener's granulomatosis -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs1805010, rs1805011, rs1805012, rs1805015, rs1801275     and rs1805016. -   21. The method of any preceding clause, wherein said antibody or     antibody fragment is administered by inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation. -   22. The method of any preceding clause, wherein the VH domain     comprises the amino acid sequence of SEQ ID NO: 69.

Ligands with VH Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of an IL4Ra-mediated disease     or condition (eg, asthma) in a human in need thereof, wherein the     ligand specifically binds a human IL4RA protein that comprises a     mutation selected from the group consisting of I75V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67; wherein (i) the ligand     comprises a VH domain derived from the recombination of a human VH     segment, a human D gene segment and a human JH segment, the human VH     segment encoding the framework 1 of SEQ ID NO: 40 and wherein said     human comprises a VH gene segment encoding the framework 1 of SEQ ID     NO: 40, or the human expresses VH domains that comprise the     framework 1 of SEQ ID NO: 40; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said IL4RA protein comprising said mutation selected from the         group consisting of I75V, E400A, C431R, S503P, Q576R and S752A         in SEQ ID NO: 67.

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting IL4Ra in a human, wherein the ligand specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;

wherein (i) the ligand comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and

wherein (ii) said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of 175V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In an example, the human is suffering from or at risk of an IL4Ra-mediated disease or condition. In an example, the method treats or reduces the risk of an IL4Ra-mediated disease or condition in the human.

-   2. The ligand of paragraph 1, wherein the IL4Ra comprises mutations     I75V and Q576R in SEQ ID NO: 67 and optionally the disease is     asthma.

The ligand of paragraph 1 or 2, wherein the nucleotide sequence of (ii) comprises nucleotide mutation −3223T (dB SNP numbering).

The ligand of any preceding paragraph, wherein each said human VH gene segment comprises SEQ ID NO: 39.

-   3. The ligand of any preceding paragraph, wherein the VH gene     segment comprised by said human is a germline VH gene segment. -   4. The ligand of any preceding paragraph, said method comprising,     before said administering, selecting a human comprising said     nucleotide sequence of (ii), wherein the human is the human of     paragraph 1. -   5. The ligand of any preceding paragraph, wherein the human has been     determined to comprise the nucleotide sequence that encodes an IL4Ra     protein comprising said mutation selected from the group consisting     of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67     and/or an IL4Ra protein comprising said mutation selected from the     group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in     SEQ ID NO: 67. -   6. The ligand of any preceding paragraph, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes an IL4Ra protein comprising said mutation     selected from the group consisting of I75V, E400A, C431R, S503P,     Q576R and S752A in SEQ ID NO: 67 and/or (b) an IL4Ra protein     comprising said mutation said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67, optionally, wherein the determining step is performed before     administration of the antibody to the human. -   7. The ligand of paragraph 8, wherein the step of determining     comprises assaying a biological sample from the human for a     nucleotide sequence encoding a IL4Ra protein comprising said     mutation selected from the group consisting of I75V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67. -   8. The ligand of paragraph 9, wherein the assaying comprises     contacting the biological sample with at least one oligonucleotide     probe comprising a sequence of at least 10 contiguous nucleotides of     a nucleotide sequence encoding an IL4Ra protein comprising said     mutation selected from the group consisting of I75V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67 or comprising an antisense     sequence of said contiguous nucleotides, wherein said sequence of     contiguous nucleotides comprises a nucleotide sequence encoding said     mutation selected from the group consisting of I75V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67, thereby forming a complex     when at least one nucleotide sequence encoding the IL4Ra protein     comprising said mutation selected from the group consisting of I75V,     E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 is present;     and detecting the presence or absence of the complex, wherein     detecting the presence of the complex determines that the human     comprises the IL4Ra protein comprising said mutation selected from     the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A     in SEQ ID NO: 67. -   9. The ligand of paragraph 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   10. The ligand of paragraph 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   11. The ligand of any preceding paragraph, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     IL4Ra protein comprising said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67, optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 68, or said human     is indicated as homozygous for a nucleotide sequence encoding the     IL4Ra protein comprising said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67. -   12. The ligand of any preceding paragraph, wherein said human is or     has been further determined to be substantially resistant to an     asthma treatment. -   13. The ligand of any preceding paragraph, wherein said human is     receiving or has received an asthma treatment or has reduced     responsiveness to an asthma treatment. -   14. The ligand of any preceding paragraph, wherein said disease or     condition is an inflammatory disease or condition; an atopic disease     or condition; a respiratory disease or condition; a disease or     condition associated with elevated IgE; or a disease or condition     associated with elevated IL-4 and/or IL-13 activity. -   15. The ligand of any preceding paragraph, wherein said disease or     condition is selected from the group consisting of an airway     inflammatory disease or condition, chronic obstructive pulmonary     disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary     infiltrate with eosinophilia, environmental lung disease, pneumonia,     bronchiectasis, cystic fibrosis, interstitial lung disease, primary     pulmonary hypertension, pulmonary thromboembolism, disorders of the     pleura, disorders of the mediastinum, disorders of the diaphragm,     hypoventilation, hyperventilation, sleep apnea, acute respiratory     distress syndrome, mesothelioma, sarcoma, graft rejection, graft     versus host disease, lung cancer, allergic rhinitis, allergy,     asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic     bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary     fibrosis, invasive pneumococcal disease, influenza, nontuberculous     mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,     pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,     pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary     inflammation, pulmonary histiocytosis X, pulmonary hypertension,     pulmonary nocardiosis, pulmonary tuberculosis, pulmonary     veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and     Wegener's granulomatosis. -   16. The ligand of any preceding paragraph, wherein said human has     been diagnosed with at least one condition selected from the group     consisting of an airway inflammatory disease or condition, chronic     obstructive pulmonary disease, asthma, pneumonia, hypersensitivity     pneumonitis, pulmonary infiltrate with eosinophilia, environmental     lung disease, pneumonia, bronchiectasis, cystic fibrosis,     interstitial lung disease, primary pulmonary hypertension, pulmonary     thromboembolism, disorders of the pleura, disorders of the     mediastinum, disorders of the diaphragm, hypoventilation,     hyperventilation, sleep apnea, acute respiratory distress syndrome,     mesothelioma, sarcoma, graft rejection, graft versus host disease,     lung cancer, allergic rhinitis, allergy, asbestosis, aspergilloma,     aspergillosis, bronchiectasis, chronic bronchitis, emphysema,     eosinophilic pneumonia, idiopathic pulmonary fibrosis, invasive     pneumococcal disease, influenza, nontuberculous mycobacteria,     pleural effusion, pneumoconiosis, pneumocytosis, pneumonia,     pulmonary actinomycosis, pulmonary alveolar proteinosis, pulmonary     anthrax, pulmonary edema, pulmonary embolus, pulmonary inflammation,     pulmonary histiocytosis X, pulmonary hypertension, pulmonary     nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive     disease, rheumatoid lung disease, sarcoidosis, and Wegener's     granulomatosis. -   17. The ligand of any preceding paragraph, wherein said antibody or     antibody fragment treats or reduces the risk in said human of at     least one condition selected from the group consisting of an airway     inflammatory disease or condition, chronic obstructive pulmonary     disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary     infiltrate with eosinophilia, environmental lung disease, pneumonia,     bronchiectasis, cystic fibrosis, interstitial lung disease, primary     pulmonary hypertension, pulmonary thromboembolism, disorders of the     pleura, disorders of the mediastinum, disorders of the diaphragm,     hypoventilation, hyperventilation, sleep apnea, acute respiratory     distress syndrome, mesothelioma, sarcoma, graft rejection, graft     versus host disease, lung cancer, allergic rhinitis, allergy,     asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic     bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary     fibrosis, invasive pneumococcal disease, influenza, nontuberculous     mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,     pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,     pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary     inflammation, pulmonary histiocytosis X, pulmonary hypertension,     pulmonary nocardiosis, pulmonary tuberculosis, pulmonary     veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and     Wegener's granulomatosis -   18. The ligand of any preceding paragraph, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs1805010, rs1805011, rs1805012, rs1805015, rs1801275     and rs1805016. -   19. The ligand of any preceding paragraph, wherein said antibody or     antibody fragment is for inhaled, intravenous or subcutaneous     administration and/or is comprised in an inhalable or injectable     preparation. -   20. The ligand of any preceding paragraph, wherein the VH domain     comprises the amino acid sequence of SEQ ID NO: 69.

Methods with C Region Tailoring

-   1. A method of treating or reducing the risk of an IL4Ra-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a human IL4Ra protein     that comprises a mutation selected from the group consisting of     I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;     -   wherein the ligand comprises a human gamma-4 heavy chain         constant region that comprises a Leu at position 189 shown in         SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73         and wherein said human comprises (i) an IGHG4*01 human heavy         chain constant region gene segment, or the human expresses         antibodies comprising human gamma-4 heavy chain constant regions         comprising a Leu at position 189 shown in SEQ ID NO: 73 or an         Arg at position 289 shown in SEQ ID NO: 73; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said IL4RA protein comprising said mutation selected from the         group consisting of I75V, E400A, C431R, S503P, Q576R and S752A         in SEQ ID NO: 67.

In an alternative, clause 1 provides:—

A method of targeting IL4Ra in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human IL4Ra protein that comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;

wherein the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In an example, the human is suffering from or at risk of an IL4Ra-mediated disease or condition. In an example, the method treats or reduces the risk of an IL4Ra-mediated disease or condition in the human.

In an example of the method or ligand of the invention, the IL4Ra comprises mutation 175V. In an example of the method or ligand of the invention, the IL4Ra comprises mutation E400A. In an example of the method or ligand of the invention, the IL4Ra comprises mutation C431R. In an example of the method or ligand of the invention, the IL4Ra comprises mutation S503P. In an example of the method or ligand of the invention, the IL4Ra comprises mutation Q576R. In an example of the method or ligand of the invention, the IL4Ra comprises mutation S752A.

-   2. The method of clause 1, wherein the IL4Ra comprises mutations     I75V and Q576R in SEQ ID NO: 67 and optionally the disease is     asthma. -   3. The method of clause 1 or 2, wherein the nucleotide sequence     of (ii) comprises nucleotide mutation −3223T (dB SNP numbering). -   4. The method of any preceding clause, wherein said human gamma-4     heavy chain constant region that comprises SEQ ID NO: 73. -   5. The method of any preceding clause, wherein the human expresses     antibodies comprising human IGHG4*01 heavy chain constant regions. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes an IL4Ra     protein comprising said mutation selected from the group consisting     of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67     and/or an IL4Ra protein comprising said mutation selected from the     group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in     SEQ ID NO: 67. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes an IL4Ra protein comprising said mutation selected from     the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A     in SEQ ID NO: 67 and/or (b) an IL4Ra protein comprising said     mutation said mutation selected from the group consisting of I75V,     E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, optionally,     wherein the determining step is performed before administration of     the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a IL4Ra protein comprising said mutation selected     from the group consisting of I75V, E400A, C431R, S503P, Q576R and     S752A in SEQ ID NO: 67. -   10. The method of clause 9, wherein the assaying comprises     -   contacting the biological sample with at least one         oligonucleotide probe comprising a sequence of at least 10         contiguous nucleotides of a nucleotide sequence encoding an         IL4Ra protein comprising said mutation selected from the group         consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ         ID NO: 67 or comprising an antisense sequence of said contiguous         nucleotides, wherein said sequence of contiguous nucleotides         comprises a nucleotide sequence encoding said mutation selected         from the group consisting of I75V, E400A, C431R, S503P, Q576R         and S752A in SEQ ID NO: 67, thereby forming a complex when at         least one nucleotide sequence encoding the IL4Ra protein         comprising said mutation selected from the group consisting of         I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 is         present; and     -   detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the IL4Ra protein comprising said mutation selected         from the group consisting of I75V, E400A, C431R, S503P, Q576R         and S752A in SEQ ID NO: 67. -   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     IL4Ra protein comprising said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67, optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 68, or said human     is indicated as homozygous for a nucleotide sequence encoding the     IL4Ra protein comprising said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to an asthma     treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received an asthma treatment or has reduced     responsiveness to an asthma treatment. -   16. The method of any preceding clause, wherein said disease or     condition is an inflammatory disease or condition; an atopic disease     or condition; a respiratory disease or condition; a disease or     condition associated with elevated IgE; or a disease or condition     associated with elevated IL-4 and/or IL-13 activity. -   17. The method of any preceding clause, wherein said disease or     condition is selected from the group consisting of an airway     inflammatory disease or condition, chronic obstructive pulmonary     disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary     infiltrate with eosinophilia, environmental lung disease, pneumonia,     bronchiectasis, cystic fibrosis, interstitial lung disease, primary     pulmonary hypertension, pulmonary thromboembolism, disorders of the     pleura, disorders of the mediastinum, disorders of the diaphragm,     hypoventilation, hyperventilation, sleep apnea, acute respiratory     distress syndrome, mesothelioma, sarcoma, graft rejection, graft     versus host disease, lung cancer, allergic rhinitis, allergy,     asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic     bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary     fibrosis, invasive pneumococcal disease, influenza, nontuberculous     mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,     pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,     pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary     inflammation, pulmonary histiocytosis X, pulmonary hypertension,     pulmonary nocardiosis, pulmonary tuberculosis, pulmonary     veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and     Wegener's granulomatosis. -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition selected from the group     consisting of an airway inflammatory disease or condition, chronic     obstructive pulmonary disease, asthma, pneumonia, hypersensitivity     pneumonitis, pulmonary infiltrate with eosinophilia, environmental     lung disease, pneumonia, bronchiectasis, cystic fibrosis,     interstitial lung disease, primary pulmonary hypertension, pulmonary     thromboembolism, disorders of the pleura, disorders of the     mediastinum, disorders of the diaphragm, hypoventilation,     hyperventilation, sleep apnea, acute respiratory distress syndrome,     mesothelioma, sarcoma, graft rejection, graft versus host disease,     lung cancer, allergic rhinitis, allergy, asbestosis, aspergilloma,     aspergillosis, bronchiectasis, chronic bronchitis, emphysema,     eosinophilic pneumonia, idiopathic pulmonary fibrosis, invasive     pneumococcal disease, influenza, nontuberculous mycobacteria,     pleural effusion, pneumoconiosis, pneumocytosis, pneumonia,     pulmonary actinomycosis, pulmonary alveolar proteinosis, pulmonary     anthrax, pulmonary edema, pulmonary embolus, pulmonary inflammation,     pulmonary histiocytosis X, pulmonary hypertension, pulmonary     nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive     disease, rheumatoid lung disease, sarcoidosis, and Wegener's     granulomatosis. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of at     least one condition selected from the group consisting of an airway     inflammatory disease or condition, chronic obstructive pulmonary     disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary     infiltrate with eosinophilia, environmental lung disease, pneumonia,     bronchiectasis, cystic fibrosis, interstitial lung disease, primary     pulmonary hypertension, pulmonary thromboembolism, disorders of the     pleura, disorders of the mediastinum, disorders of the diaphragm,     hypoventilation, hyperventilation, sleep apnea, acute respiratory     distress syndrome, mesothelioma, sarcoma, graft rejection, graft     versus host disease, lung cancer, allergic rhinitis, allergy,     asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic     bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary     fibrosis, invasive pneumococcal disease, influenza, nontuberculous     mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,     pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,     pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary     inflammation, pulmonary histiocytosis X, pulmonary hypertension,     pulmonary nocardiosis, pulmonary tuberculosis, pulmonary     veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and     Wegener's granulomatosis -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs1805010, rs1805011, rs1805012, rs1805015, rs1801275     and rs1805016. -   21. The method of any preceding clause, wherein said antibody or     antibody fragment is administered by inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation.

Ligands with Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of an IL4Ra-mediated disease     or condition (eg, asthma) in a human in need thereof, wherein the     ligand specifically binds a human IL4RA protein that comprises a     mutation selected from the group consisting of 175V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67;     -   wherein (i) the ligand comprises a human gamma-4 heavy chain         constant region that comprises a Leu at position 189 shown in         SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73         and wherein said human comprises (i) an IGHG4*01 human heavy         chain constant region gene segment, or the human expresses         antibodies comprising human gamma-4 heavy chain constant regions         comprising a Leu at position 189 shown in SEQ ID NO: 73 or an         Arg at position 289 shown in SEQ ID NO: 73; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said IL4RA protein comprising said mutation selected from the         group consisting of I75V, E400A, C431R, S503P, Q576R and S752A         in SEQ ID NO: 67.

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting IL4Ra in a human, wherein the ligand specifically binds a human IL4RA protein that comprises a mutation selected from the group consisting of 175V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;

wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Len at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said IL4RA protein comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In an example, the human is suffering from or at risk of an IL4Ra-mediated disease or condition. In an example, the method treats or reduces the risk of an IL4Ra-mediated disease or condition in the human.

-   2. The ligand of paragraph 1, wherein the IL4Ra comprises mutations     175V and Q576R in SEQ ID NO: 67 and optionally the disease is     asthma. -   3. The ligand of paragraph 1 or 2, wherein the nucleotide sequence     of (ii) comprises nucleotide mutation −3223T (dB SNP numbering). -   4. The ligand of any preceding paragraph, wherein said human gamma-4     heavy chain constant region that comprises SEQ ID NO: 73. -   5. The ligand of any preceding paragraph, wherein the human     expresses antibodies comprising human IGHG4*01 heavy chain constant     regions. -   6. The ligand of any preceding paragraph, said method comprising,     before said administering, selecting a human comprising said     nucleotide sequence of (ii), wherein the human is the human of     paragraph 1. -   7. The ligand of any preceding paragraph, wherein the human has been     determined to comprise the nucleotide sequence that encodes an IL4Ra     protein comprising said mutation selected from the group consisting     of 175V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67     and/or an IL4Ra protein comprising said mutation selected from the     group consisting of 175V, E400A, C431R, S503P, Q576R and S752A in     SEQ ID NO: 67. -   8. The ligand of any preceding paragraph, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes an IL4Ra protein comprising said mutation     selected from the group consisting of I75V, E400A, C431R, S503P,     Q576R and S752A in SEQ ID NO: 67 and/or (b) an IL4Ra protein     comprising said mutation said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67, optionally, wherein the determining step is performed before     administration of the antibody to the human. -   9. The ligand of paragraph 8, wherein the step of determining     comprises assaying a biological sample from the human for a     nucleotide sequence encoding a IL4Ra protein comprising said     mutation selected from the group consisting of I75V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67. -   10. The ligand of paragraph 9, wherein the assaying comprises     contacting the biological sample with at least one oligonucleotide     probe comprising a sequence of at least 10 contiguous nucleotides of     a nucleotide sequence encoding an IL4Ra protein comprising said     mutation selected from the group consisting of I75V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67 or comprising an antisense     sequence of said contiguous nucleotides, wherein said sequence of     contiguous nucleotides comprises a nucleotide sequence encoding said     mutation selected from the group consisting of I75V, E400A, C431R,     S503P, Q576R and S752A in SEQ ID NO: 67, thereby forming a complex     when at least one nucleotide sequence encoding the IL4Ra protein     comprising said mutation selected from the group consisting of I75V,     E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 is present;     and detecting the presence or absence of the complex, wherein     detecting the presence of the complex determines that the human     comprises the IL4Ra protein comprising said mutation selected from     the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A     in SEQ ID NO: 67. -   11. The ligand of paragraph 9, wherein the assaying comprises     nucleic acid amplification and optionally one or more methods     selected from sequencing, next generation sequencing, nucleic acid     hybridization, and allele-specific amplification and/or wherein the     assaying is performed in a multiplex format. -   12. The ligand of paragraph 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding paragraph, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     IL4Ra protein comprising said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67, optionally, wherein said human is further indicated as     comprising the nucleotide sequence of SEQ ID NO: 68, or said human     is indicated as homozygous for a nucleotide sequence encoding the     IL4Ra protein comprising said mutation selected from the group     consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID     NO: 67. -   14. The ligand of any preceding paragraph, wherein said human is or     has been further determined to be substantially resistant to an     asthma treatment. -   15. The ligand of any preceding paragraph, wherein said human is     receiving or has received an asthma treatment or has reduced     responsiveness to an asthma treatment. -   16. The ligand of any preceding paragraph, wherein said disease or     condition is an inflammatory disease or condition; an atopic disease     or condition; a respiratory disease or condition; a disease or     condition associated with elevated IgE; or a disease or condition     associated with elevated IL-4 and/or IL-13 activity. -   17. The ligand of any preceding paragraph, wherein said disease or     condition is selected from the group consisting of an airway     inflammatory disease or condition, chronic obstructive pulmonary     disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary     infiltrate with eosinophilia, environmental lung disease, pneumonia,     bronchiectasis, cystic fibrosis, interstitial lung disease, primary     pulmonary hypertension, pulmonary thromboembolism, disorders of the     pleura, disorders of the mediastinum, disorders of the diaphragm,     hypoventilation, hyperventilation, sleep apnea, acute respiratory     distress syndrome, mesothelioma, sarcoma, graft rejection, graft     versus host disease, lung cancer, allergic rhinitis, allergy,     asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic     bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary     fibrosis, invasive pneumococcal disease, influenza, nontuberculous     mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,     pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,     pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary     inflammation, pulmonary histiocytosis X, pulmonary hypertension,     pulmonary nocardiosis, pulmonary tuberculosis, pulmonary     veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and     Wegener's granulomatosis. -   18. The ligand of any preceding paragraph, wherein said human has     been diagnosed with at least one condition selected from the group     consisting of an airway inflammatory disease or condition, chronic     obstructive pulmonary disease, asthma, pneumonia, hypersensitivity     pneumonitis, pulmonary infiltrate with eosinophilia, environmental     lung disease, pneumonia, bronchiectasis, cystic fibrosis,     interstitial lung disease, primary pulmonary hypertension, pulmonary     thromboembolism, disorders of the pleura, disorders of the     mediastinum, disorders of the diaphragm, hypoventilation,     hyperventilation, sleep apnea, acute respiratory distress syndrome,     mesothelioma, sarcoma, graft rejection, graft versus host disease,     lung cancer, allergic rhinitis, allergy, asbestosis, aspergilloma,     aspergillosis, bronchiectasis, chronic bronchitis, emphysema,     eosinophilic pneumonia, idiopathic pulmonary fibrosis, invasive     pneumococcal disease, influenza, nontuberculous mycobacteria,     pleural effusion, pneumoconiosis, pneumocytosis, pneumonia,     pulmonary actinomycosis, pulmonary alveolar proteinosis, pulmonary     anthrax, pulmonary edema, pulmonary embolus, pulmonary inflammation,     pulmonary histiocytosis X, pulmonary hypertension, pulmonary     nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive     disease, rheumatoid lung disease, sarcoidosis, and Wegener's     granulomatosis. -   19. The ligand of any preceding paragraph, wherein said antibody or     antibody fragment treats or reduces the risk in said human of at     least one condition selected from the group consisting of an airway     inflammatory disease or condition, chronic obstructive pulmonary     disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary     infiltrate with eosinophilia, environmental lung disease, pneumonia,     bronchiectasis, cystic fibrosis, interstitial lung disease, primary     pulmonary hypertension, pulmonary thromboembolism, disorders of the     pleura, disorders of the mediastinum, disorders of the diaphragm,     hypoventilation, hyperventilation, sleep apnea, acute respiratory     distress syndrome, mesothelioma, sarcoma, graft rejection, graft     versus host disease, lung cancer, allergic rhinitis, allergy,     asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic     bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary     fibrosis, invasive pneumococcal disease, influenza, nontuberculous     mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,     pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,     pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary     inflammation, pulmonary histiocytosis X, pulmonary hypertension,     pulmonary nocardiosis, pulmonary tuberculosis, pulmonary     veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and     Wegener's granulomatosis -   20. The ligand of any preceding paragraph, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs1805010, rs1805011, rs1805012, rs1805015, rs1801275     and rs1805016. -   21. The ligand of any preceding paragraph, wherein said antibody or     antibody fragment is for inhaled, intravenous or subcutaneous     administration and/or is comprised in an inhalable or injectable     preparation.

In an example of any other example or embodiment, clause or paragraph relating to IL4Ra, the IL4Ra mutation is 75V and the human is of AFR or ASN ancestry, eg, the ancestry of the human is selected from ASW, LWK, CHS or JPT. All of these ancestries have greater than the average of 47% frequency for 75V.

In an example of any other example or embodiment, clause or paragraph relating to IL4Ra, the IL4Ra mutation is 400A and the human is of AFR ancestry, eg, the ancestry of the human is selected from ASW, LWK and YRJ. All of these ancestries have greater than the average frequency for 400A.

In an example of any other example or embodiment, clause or paragraph relating to IL4Ra, the IL4Ra mutation is 431R and the human is of AFR or AMR ancestry, eg, the ancestry of the human is selected from YRI, CLM, MXL, PUR or CEU. All of these ancestries have greater than the average frequency for 431R.

In an example of any other example or embodiment, clause or paragraph relating to IL4Ra, the IL4Ra mutation is 752A and the human is of AFR ancestry, eg, the ancestry of the human is selected from ASW, LWK and YRI. All of these ancestries have 37-39% frequency, which is greater than the average frequency for 752A.

Frequencies are with reference to the 1000 Genomes database (version 20110521) here.

In an example of any other example or embodiment, clause or paragraph relating to IL4Ra, the IL4Ra mutation is 75V and the human is of AFR or ASN ancestry, eg, the ancestry of the human is selected from ASW, LWK, CHS or JPT. All of these ancestries have greater than the average of 47% frequency for 75V.

Example 3: Nav1.7 (SCN9A; ETHA; FEB3B; GEFSP7; NE-NA; NENA; PN1; SFNP)

Reference is made to J Clin Invest. 2007 December; 117(12):3603-9, “Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders”, Drenth J P & Waxman S G (incorporated herein by reference). Voltage-gated sodium channels play a critical role in the generation and conduction of action potentials and are thus important for electrical signalling by most excitable cells. Sodium channels are integral membrane proteins and are comprised of a large a subunit, which forms the voltage-sensitive and ion-selective pore, and smaller auxiliary $ subunit(s) that can modulate the kinetics and voltage dependence of channel gating. To date, we know of 9 isoforms of the sodium-channel a subunit (Nav1.1-Nav1.9), each with a unique central and peripheral nervous system distribution. Four closely related sodium channels (Nav1.1, -1.2, -1.3, and -1.7) are encoded by a set of 4 genes (SCN1A, SCN2A, SCN3A, and SCN9A, respectively) located within a cluster on chromosome 2q24.3. Nav1.7 has a critical role in pain sensation. Furthermore, from KO studies and animal pain models, it would appear that Nav1.7 plays a major role in inflammatory pain.

Nav1.7 is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Nav1.7 dysfunction in three different human pain disorders. Gain-of-function missense mutations in Nav1.7 have been shown to cause primary erythermalgia (PE) and paroxysmal extreme pain disorder (PEPD), while nonsense mutations in Nav1.7 result in loss of Nav1.7 function and a condition known as channelopathy-associated insensitivity to pain (CIP), a rare disorder in which affected individuals are unable to feel physical pain. Recent work has shown that different types of channelopathies (diseases caused by disturbed function of ion channel subunits or the proteins that regulate them), all involving the same Nav1.7 sodium channel, underlie these disorders.

Nav1.7 is encoded by SCN9A, a 113.5-kb gene comprising 26 exons (OMIM 603415) (FIG. 1A, Drenth & Waxman). The encoded sodium channel is composed of 1977 amino acids organized into 4 domains, each with 6 transmembrane segments, and is predominantly expressed in the dorsal root ganglion (DRG) neurons and sympathetic ganglion neurons (FIG. 1B, Drenth & Waxman). Immunohistochemical studies show that Nav1.7 is present at the distal ends of the wire-like projections of neurons known as neurites, close to the impulse trigger zone where neuronal firing is initiated. Interestingly, the large majority of DRG neurons that express Nav1.7 are pain sensing (nociceptive), suggesting a role for this sodium channel in the pathogenesis of pain.

Cell. 2014 Jun. 5; 157(6):1393-404. doi: 10.1016/j.cell.2014.03.064. Epub 2014 May 22; “A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief”, Lee J H et al and 2011 (both incorporated herein by reference) describe the generation of a monoclonal antibody against huma Nav1.7.

In an example, the invention provides a method of treating or reducing the risk of a Nav1.7-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a huma Nav1.7 protein. The invention also provides a corresponding ligand.

The present invention provides anti-Nav1.7 ligands; and Nav1.7-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of Nav1.7, in particular huma Nav1.7 or its ligands and in screening assays to identify other antagonists of Nav1.7 activity. Some of the ligands of the invention are useful for inhibiting Nav1.7-mediated activities.

Anti-Nav1.7 ligands (eg, antibodies and anti-sense RNA) have been developed based on targeting and neutralising so-called “wild-type” huma Nav1.7, which is a commonly-occurring form (see, eg, SEQ ID NO: 75). While such therapies are useful for human patients harbouring this form of huma Nav1.7, the inventor considered it useful to investigate the possibility of targeting rarer—but still naturally-occurring—forms of Nav1.7 amongst human populations. In this way, the inventor arrived at insight into the natural occurrences and distributions of rarer huma Nav1.7 forms that can serve as useful targets (at the protein or nucleic acid level) for human treatment, prophylaxis and diagnosis pertinent to diseases and conditions mediated or associated with NAV1.7 activity. This particularly provides for tailored therapies, prophylaxis and diagnosis in humans that are devoid of the common Nav1.7 gene or protein.

The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in activity and/or conformation of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to more effectively tailor medicines and diagnosis of patients. The invention, therefore, provides for tailored pharmaceuticals and testing that specifically addresses rarer Nav1.7 polymorphic variant forms. Such forms or “alleles” (at the nucleotide level), comprise one or more changes at the nucleotide and amino acid levels from the corresponding common form nucleotide and amino acids sequences, ie, there are one or more non-synonymous (aka “missense”) changes at the nucleotide level that translate into one or more corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting such rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention.

With this realisation, the inventor saw that there is significant industrial and medical application for the invention in terms of guiding the choice of anti-Nav1.7 ligand for administration to human patients for therapy and/or prophylaxis of Nav1.7-mediated or associated diseases or conditions. In this way, the patient receives drugs and ligands that are tailored to their needs—as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example the present inventor decided to determine a set of huma Nav1.7 variants on the basis of the following criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population. The inventor selected variants having at least 3 of the 4 following criteria:—

Naturally-occurring huma Nav1.7 variation having a cumulative human allele frequency of 35% or less;

Naturally-occurring huma Nav1.7 variation having a total human genotype frequency of about 50% or less;

Naturally-occurring huma Nav1.7 variation found in many different human ethnic populations (using the standard categorisation of the 1000 Genomes Project; see Table 2 below); and

Naturally-occurring huma Nav1.7 variation found in many individuals distributed across such many different ethnic populations.

On the basis of these criteria, the inventor identified variants listed in Table 12 (see Further Variants) below. The inventor's selection included, as an additional or alternative consideration, selection for nucleotide variation that produced amino acid variation in corresponding Nav1.7 forms (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.

In an example, the invention provides a method of treating or reducing the risk of a Nav1.7-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. As explained further below, these amino acid variations are found in naturally-occurring Nav1.7 variants in humans found in many populations. Said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

For example, said amino acid is selected from the group consisting of any one of (a) to (e):—

-   -   a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; or     -   b) 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; or     -   c) 277X (wherein X is an amino acid other than R), 328X (wherein         X is an amino acid other than Y), 395K, 459X (wherein X is an         amino acid other than S), 693 X (wherein X is an amino acid         other than E), 767X (wherein X is an amino acid other than I),         830X (wherein X is an amino acid other than R), 897X (wherein X         is an amino acid other than W), 1200L, 1235L, 1488X (wherein X         is an amino acid other than R), 1659X (wherein X is an amino         acid other than K) and 1689X (wherein X is an amino acid other         than W); or     -   d) 395K, 1200L and 1235L; or     -   c) 422D, 490N, 943L, 1002L, 1161W and 1919G.         Amino acids (a)-(d) are discussed in Drenth & Waxman as being         associated with pain conditions (either undesirable elevated or         reduced pain). Thus, in one embodiment, the disease or condition         is a pain disease or condition and the amino acid is selected         from (a), (b), (c) or (d). Thus, in one embodiment, the disease         or condition is an itching disease or condition and the amino         acid is selected from (a), (b), (c) or (d). In an example, the         disease is PE and the amino acid is selected from (a). In an         example, the disease is PEPD and the amino acid is selected from         (b). In an example, the disease is CIP and the amino acid is         selected from (c) or (d). In an example, the disease or         condition is a pain or itching disease or condition and the         amino acid is selected from (e).

In one embodiment, the pain is chronic pain.

In one embodiment, the pain is neuropathic pain, eg, chronic neuropathic pain. For example, the pain is painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN) or trigeminal neuralgia (TN).

In an example, the pain is spinal cord injury pain, multiple sclerosis pain, phantom limb pain, post-stroke pain, chronic back pain, osteoarthritis pain, cancer-associated pain or HIV-associated pain.

In one embodiment, the pain is inflammatory pain, eg, chronic inflammatory pain.

In one embodiment, the disease or condition is a channelopathy or associated with a channelopathy.

In one embodiment, the disease or condition is selected from the group consisting of primary erythermalgia (PE), paroxysmal extreme pain disorder (PEPD) and channelopathy-associated insensitivity to pain (CIP).

Optionally, the NAV-mediated disease or condition is selected from any one of the following:

-   -   a. Neuropathic or neurogenic pain (for example arising from         painful diabetic neuropathy (PDN), post-herpetic neuropathy         (PHN), central neuropathy, peripheral neuropathy, trigeminal         neuralgia (TN), anaesthesia dolorosa, spinal cord injuries,         multiple sclerosis, phantom limb pain, hyperalgesia,         hyperpathia, paresthesia, psychogenic pain, post-stroke pain and         HIV-associated pain, back pain, chronic back pain,         osteoarthritis, cancer, breakthrough pain, erythromelalgia [e.g.         primary erythromelalgia], paroxysmal 30 extreme pain disorder,         nerve compression and/or entrapment [such as carpal tunnel         syndrome, tarsal tunnel syndrome, ulnar nerve entrapment,         compression radiculopathy, radicular low back pain, spinal root         lesions, spinal root compression, lumbar spinal stenosis,         sciatic nerve compression, intercostal neuralgia], neuritis,         pain from chemotherapy, congenital defect/channelopathy [e.g.         channelopathy-associated insensitivity to pain and congenital         insensitivity to pain] or chronic alcoholism [alcoholic         polyneuropathy]);     -   b. inflammatory pain (such as osteoarthritis pain, chronic back         pain, rheumatoid arthritis pain, cancer pain, breakthrough pain,         pain associated with burns, encephalitis pain, bone fracture         pain, neuritis pain, autoimmune disease pain, postoperative         pain, dental pain, pain associated with bacterial infection,         pain associated with radiotherapy, pain associated with gout and         irritable bowel syndrome pain);     -   c. pain from trauma (such as from lacerations, incisions, burns,         foreign bodies or bullet and/or shrapnel injuries, spinal cord         injury, brachial plexus avulsion, nerve crush and/or entrapment         (such as carpal tunnel syndrome, tarsal tunnel syndrome, ulnar         nerve entrapment, compression radiculopathy, radicular low back         pain, spinal root lesions, spinal root compression, lumbar         spinal stenosis, sciatic nerve compression, intercostal         neuralgia), nerve transection, post-operative pain, dental pain         and toxic exposure);     -   d. pain from infection (such as post-herpetic neuropathy (PHN),         HIV-associated pain small pox infection, encephalitis, herpes         infection, and bacterial infection);     -   e. pain from malignancy (such as cancer pain, breakthrough pain,         and nerve compression pain);     -   f. visceral pain (such as renal or ureteral colic, irritable         bowel syndrome, angina or cardiac pain, cardiac arrhythmia,         period pain, interstitial cystitis, rectal pain, pain associated         with diarrhoea, appendicitis, cholecystitis and pancreatitis);     -   g. metabolic or chronic disease pain (such as associated with         multiple sclerosis, cancer pain, breakthrough pain, pain         associated with gout, peripheral diabetic neuropathy, pain         associated with chronic alcoholism [alcoholic polyneuropathy],         uremia pain, pain associated with hypothyroidism and pain         associated with vitamin deficiency);     -   h. headache pain (such as tension headache, migraine and cluster         headache);     -   i. idiopathic pain (such as trigeminal neuralgia, a complex         regional pain syndrome [e.g. complex regional pain syndrome I         and/or complex regional pain syndrome II], allodynia or         fibromyalgia);     -   j. respiratory pain (such as pain associated with asthma, airway         hyper-reactivity in asthma, chronic cough, e.g. in asthma and/or         chronic obstructive pulmonary disorder); or     -   k. other pain (such as pain associated with hormonal therapy,         diabetes, hypothyroidism, epilepsy, ataxia, periodic paralysis,         acute itch or chronic itch).

In an example, the NAV-mediated disease or condition is selected from painful diabetic neuropathy, post-herpetic neuropathy, trigeminal neuralgia, osteoarthritis, chronic back pain, nerve compression pain (e.g. sciatic nerve compression) or cancer pain; or is selected from migraine, post-operative pain and fibromyalgia.

In an example, the ligand of the invention comprises an anti-huma Nav1.7 binding site, wherein the binding site is a human or humanized binding site, eg, the binding site comprises or consists of a human or humanized antibody variable domain or plurality of variable domains (eg, human VH/VL binding site(s)). Additionally or alternatively, the ligand comprises one or more human antibody constant regions (eg, a human antibody CH1, CH2, CH3 (or all of these) or Fc). In an example, the ligand is an antibody that comprises human or humanized variable regions and human constant regions (eg, bearing one or more mutations to enhance or dampen Fc function in a human patient).

An example provides a ligand (eg, an antibody or antibody fragment) for treating or reducing the risk of a Nav1.7-mediated disease or condition in a human in need thereof, the method comprising administering to said human said ligand, wherein the ligand specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the invention provides a method of targeting Nav1.7 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. In an example, the human is suffering from or at risk of a Nav1.7-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.7-mediated disease or condition in the human.

An example also provides a ligand (eg, an antibody or antibody fragment) for targeting Nav1.7 in a human, the method comprising administering to said human said ligand, wherein the ligand specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. In an example, the human is suffering from or at risk of a Nav1.7-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.7-mediated disease or condition in the human.

In an embodiment, (i) the antibody or fragment comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

Additionally or alternatively, in an embodiment, (i) the antibody or fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the ligand, antibody or fragment is for treating or reducing the risk of (or treats or reduces the risk of) pain or itching, optionally wherein the antibody is a humanized mouse or Camelid (eg, llama or camel) antibody.

In a specific embodiment, the anti-Nav1.7 ligand, antibody or fragment also specifically binds to another Nav selected from Nav1.1-1.9, eg, it specifically binds to Nav1.8 or 1.9.

In a specific embodiment, the anti-Nav1.7 ligand, antibody or fragment of present invention comprises an Fc region, wherein the Fc region comprises at least one non-native amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may comprise additional and/or alternative non-native amino acid residues known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

The ligand, antibody or fragment according to the invention is for treating or preventing or reducing the risk of (or treats or prevents or reduces the risk of), for example, any disease or condition disclosed in WO2011/051351, the disclosure of which diseases and conditions are incorporated herein by reference for potential inclusion in one or more claims herein. Guidance on obtaining and testing antibodies can also be found in that PCT application.

Further encompassed by the invention is the use of the ligand, antibody or fragment in the manufacture of a medicament for use to attenuate or inhibit a Nav1.7-mediated disease or disorder in a human. Nav1.7-mediated or related disorders which are treated by the ligand, antibody or fragment of the invention include, for example, a pain or itching disease or condition.

Thus, a ligand, antibody or fragment of the invention is useful as a therapeutic agent in the treatment of a condition involving Nav1.7 expression and/or activity. One embodiment, among others, is a method of treatment comprising administering an effective amount of a ligand, antibody or fragment of the invention to a patient in need thereof, wherein functional consequences of Nav1.7 activation are decreased. Another embodiment, among others, is a method of treatment comprising (i) identifying a patient demonstrating Nav1.7 expression or activity, and (ii) administering an effective amount of a ligand, antibody or fragment of the invention to the patient, wherein a functional consequence of Nav1.7 activation are attenuated. An effective amount according to the invention is an amount that modulates (e.g. decreases) the functional consequences of Nav1.7 activation so as to modulate (e.g. decrease or lessen) the severity of at least one symptom of the particular disease or disorder being treated, but not necessarily cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, comprising administering to a patient in need thereof an effective amount of one or more ligands, antibodies or fragments of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein such that the severity of at least one symptom of any of the disorders is reduced. Another embodiment of the invention, among others, is a method of antagonizing at least one effect of Nav1.7 comprising contacting with or administering an effective amount of one or more ligands, antibodies or fragments of the present invention such that said at least one effect of Nav1.7 is antagonized, e.g. the ability of Nav1.7 to form an ion channel, such as a sodium channel.

Tailoring Antibodies to Rarer Nav1.7 Variant Profile

As outlined herein (for example, in the context of PCSK9 in Example 1), the invention includes the possibility to tailor treatment of humans further by selecting antibody-based ligands with variable domains and/or constant domains based on gene segments found in many humans of the ethnic populations where the variant TOI forms are found to meet the selection criteria of the invention. This also applies mutatis mutandis where the TOI is huma Nav1.7, as in the present example. Thus, all disclosure herein relating to tailoring variable and/or constant domains apply to the present example, relating to Nav1.7 and is combinable for use in one or more claims herein.

As described in Example 1, an example is provided for ligands comprising antibody VH domains derived from recombination of human IGHV gene segments comprising selected nucleotides at positions in the HCDR1 or FW3 where there is variability in humans (ie, where SNPs occur in humans).

Further information is provided in Table 4, which shows variation at these positions, as well as the variant distributions across the 1000 Genomes Project database relating to many human populations.

In other embodiments, as explained more fully above, the invention provides for ligands which are tailored to the human recipient's genotype and/or phenotype based on alternative human VH gene segments, or on Vκ, Vλ or constant region gene segments (see further Table 9 for representative variants).

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23*04), a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40. (ii) wherein the ligand comprises a VH domain derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment, and wherein said human comprises a IGHV3-7*01 VH gene segment or the human expresses VH domains derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment. (iii) wherein the ligand comprises a Vκ domain derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment, and wherein said human comprises a IGKV1-12*01 Vκ gene segment or the human expresses Vκ domains derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment. (iv) wherein the ligand comprises a Vκ domain derived from the recombination of a human Vκ segment and a human Jκ segment, the human Vκ segment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36 and wherein said human comprises a Vκ gene segment encoding a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the human expresses Vκ domains that comprise a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 and wherein said human comprises a Vκ gene segment encoding a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 or the human expresses Vκ domains that comprise a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38. (v) wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4. (vi) wherein the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6. (vii) wherein the ligand comprises a human kappa chain constant region that comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16 and wherein said human comprises (i) an IGKC1*01 human kappa chain constant region gene segment, or the human expresses antibodies comprising human kappa chain constant regions comprising a Val corresponding to position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16. (viii) wherein the ligand comprises a human IGLC1*01 lambda chain constant region and wherein said human comprises (i) a human IGLC1*01 lambda chain constant region gene segment, or the human expresses antibodies comprising human IGLC1*01 lambda chain constant regions. (ix) wherein the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73. (x) wherein the ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3*01) constant region gene segment and wherein said human comprises (i) said first constant region gene segment (eg, an IGHG3*01), or the human expresses antibodies comprising human gamma-3 heavy chain constant regions encoded by said first human IGHG3 (eg, IGHG3*01) constant region gene segment. (xi) wherein the ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human epsilon heavy chain constant regions encoded by said first constant region gene segment. (xii) wherein the ligand comprises a human mu heavy chain constant region encoded by a first human mu heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human mu heavy chain constant regions encoded by said first constant region gene segment. (xiii) wherein the ligand comprises a human alpha heavy chain constant region encoded by a first human alpha heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human alpha heavy chain constant regions encoded by said first constant region gene segment. (xiv) wherein the ligand comprises a human delta heavy chain constant region encoded by a first human delta heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human delta heavy chain constant regions encoded by said first constant region gene segment. (xv) wherein the ligand comprises a human kappa light chain constant region encoded by a first human kappa light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human kappa light chain constant regions encoded by said first constant region gene segment. (xvi) wherein the ligand comprises a human lambda light chain constant region encoded by a first human lambda light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human lambda light chain constant regions encoded by said first constant region gene segment.

These examples, although written in the context of the present Nav1.7 example, are applicable to the invention as it relates to any other TOI herein and thus can be combined and used in claims herein relating to any TOI. In any of these example, the ligand is optionally an antibody, eg, a human or humanised antibody (eg, a humanised mouse, rat or Camelid antibody).

For ADCC efficacy for human constant regions is: IgG1≥IgG3>>IgG4≥IgG2

For CDC efficacy for human constant regions is: IgG3≥IgG1>>IgG2≈IgG4

Thus, it may be advantageous for Nav1.7 ligands, for the ligand to comprise a gamma-4 or gamma-2 constant region (eg, as per the examples above). For example, the gamma-4 is an IgG4PE (ie, a gamma-4 constant region with 228Pro and 235Glu). For example the gamma-2 is an IgG2Aa.

For example, as per example (ix), the inventor identified the possibility of addressing IGH-gamma-4 variation and identified utility for variations 189L and 289R individually or in combination, since these residues are part of the CH3 domain, and as such they form part of antibody Fc regions. Thus, matching of these CH3 variations with the patient is especially beneficial for reasons as discussed above. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human gamma-4 heavy chain constant region that comprises a Leu corresponding to position 189 of SEQ ID NO: 73 or an Arg corresponding to position 289 of SEQ ID NO: 73 and wherein the genome of the human comprises a gamma-4 heavy chain constant region nucleotide sequence that encodes such a Leu and/or Arg or the human expresses antibodies comprising human gamma-4 constant regions comprising such a Leu and/or Arg.

Determination of Specific Binding of Ligands of the Invention to NAV1.7 Variants

The specific binding of ligands of the invention to Nav1.7 variants can be performed using the SPR method described in Example 1.

Embodiments are provided as follows:—

Methods with VH Tailoring

-   1. A method of treating or reducing the risk of a Nav1.7-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a huma Nav1.7 protein     that comprises an amino acid selected from the group consisting of     136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,     1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an     amino acid other than R), 328X (wherein X is an amino acid other     than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X     (wherein X is an amino acid other than E), 767X (wherein X is an     amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;     -   wherein (i) the ligand comprises a VH domain derived from the         recombination of a human VH segment, a human D gene segment and         a human JH segment, the human VH segment encoding the framework         1 of SEQ ID NO: 40 and wherein said human comprises a VH gene         segment encoding the framework 1 of SEQ ID NO: 40, or the human         expresses VH domains that comprise the framework 1 of SEQ ID NO:         40; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.7 protein comprising said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an alternative, clause 1 provides:—

A method of targeting Nav1.7 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;

wherein (i) the ligand comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the human is suffering from or at risk of a Nav1.7-mediated disease or condition.

In an example, the method treats or reduces the risk of a Nav1.7-mediated disease or condition in the human.

-   2. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of any one of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said     disease or condition is a pain disease or condition. -   3. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I and 1627K; optionally wherein said disease or     condition is a pain disease or condition. -   4. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of 277X (wherein X is an amino     acid other than R), 328X (wherein X is an amino acid other than Y),     395K, 459X (wherein X is an amino acid other than S), 693 X (wherein     X is an amino acid other than E), 767X (wherein X is an amino acid     other than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K) and 1689X (wherein X is an amino acid other     than W); optionally wherein said disease or condition is a pain     disease or condition. -   5. The method of any preceding clause, wherein the VH gene segment     comprised by said human is a germline VH gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     and/or a Nav1.7 protein comprising said amino acid selected from the     group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,     1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X     (wherein X is an amino acid other than R), 328X (wherein X is an     amino acid other than Y), 395K, 459X (wherein X is an amino acid     other than S), 693 X (wherein X is an amino acid other than E), 767X     (wherein X is an amino acid other than I), 830X (wherein X is an     amino acid other than R), 897X (wherein X is an amino acid other     than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than     R), 1659X (wherein X is an amino acid other than K), 1689X (wherein     X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and     1919G. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes a Nav1.7 protein comprising said amino acid selected     from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,     858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,     1627K, 277X (wherein X is an amino acid other than R), 328X (wherein     X is an amino acid other than Y), 395K, 459X (wherein X is an amino     acid other than S), 693 X (wherein X is an amino acid other than E),     767X (wherein X is an amino acid other than I), 830X (wherein X is     an amino acid other than R), 897X (wherein X is an amino acid other     than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than     R), 1659X (wherein X is an amino acid other than K), 1689X (wherein     X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and     1919G and/or (b) a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G, optionally, wherein the determining step is     performed before administration of the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G. -   10. The method of clause 9, wherein the assaying comprises     contacting the biological sample with at least one oligonucleotide     probe comprising a sequence of at least 10 contiguous nucleotides of     a nucleotide sequence encoding a Nav1.7 protein comprising said     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or comprising an     antisense sequence of said contiguous nucleotides, wherein said     sequence of contiguous nucleotides comprises a nucleotide sequence     encoding said amino acid selected from the group consisting of 136I,     216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D,     1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid     other than R), 328X (wherein X is an amino acid other than Y), 395K,     459X (wherein X is an amino acid other than S), 693 X (wherein X is     an amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby forming a     complex when at least one nucleotide sequence encoding the Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     is present; and

detecting the presence or absence of the complex, wherein detecting the presence of the complex determines that the human comprises the Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

-   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.7 protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,     optionally, wherein said human is further indicated as comprising     the nucleotide sequence of SEQ ID NO: 76, or said human is indicated     as homozygous for a nucleotide sequence encoding the Nav1.7 protein     comprising said amino acid selected from the group consisting of     136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,     1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an     amino acid other than R), 328X (wherein X is an amino acid other     than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X     (wherein X is an amino acid other than E), 767X (wherein X is an     amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The method of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The method of any preceding clause, wherein said disease or     condition is a channelopathy or associated with a channelopathy; or     is selected from the group consisting of primary erythermalgia (PE),     paroxysmal extreme pain disorder (PEPD) and channelopathy-associated     insensitivity to pain (CIP). -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs6746030, rs3750904, rs58022607, rs4369876,     rs13402180 and rs12478318. -   21. The method of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is administered by inhaled,     intravenous or subcutaneous administration and/or is comprised in an     inhalable or injectable preparation.

Preferably herein the Nav1.7 mutation is 1161W (also known as 1150W in some publications, eg, Reimann et al (Proc Natl Acad Sci USA. 2010 Mar. 16; 107(11):5148-53. doi: 10.1073/pnas.0913181107. Epub 2010 Mar. 8; “Pain perception is altered by a nucleotide polymorphism in SCN9A”; Reimann F et al). This describes the correlation of 1150W (ie, 1161W) with increased pain perception (lower threshold) in normal humans.

Ligands with VH Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of a Nav1.7-mediated disease     or condition (eg, asthma) in a human in need thereof, wherein the     ligand specifically binds a human Nav1.7 protein that comprises an     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;     -   wherein (i) the ligand comprises a VH domain derived from the         recombination of a human VH segment, a human D gene segment and         a human JH segment, the human VH segment encoding the framework         1 of SEQ ID NO: 40 and wherein said human comprises a VH gene         segment encoding the framework 1 of SEQ ID NO: 40, or the human         expresses VH domains that comprise the framework 1 of SEQ ID NO:         40; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.7 protein comprising said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting Nav1.7 in a human, wherein the ligand specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;

wherein (i) the ligand comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the human is suffering from or at risk of a Nav1.7-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.7-mediated disease or condition in the human.

-   2. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of any one of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said     disease or condition is a pain disease or condition. -   3. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I and 1627K; optionally wherein said disease or     condition is a pain disease or condition. -   4. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of 277X (wherein X is an amino     acid other than R), 328X (wherein X is an amino acid other than Y),     395K, 459X (wherein X is an amino acid other than S), 693 X (wherein     X is an amino acid other than E), 767X (wherein X is an amino acid     other than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K) and 1689X (wherein X is an amino acid other     than W); optionally wherein said disease or condition is a pain     disease or condition. -   5. The ligand of any preceding clause, wherein the VH gene segment     comprised by said human is a germline VH gene segment. -   6. The ligand of any preceding clause said method comprising, before     said administering, selecting a human comprising said nucleotide     sequence of (ii), wherein the human is the human of clause 1. -   7. The ligand of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     and/or a Nav1.7 protein comprising said amino acid selected from the     group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,     1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X     (wherein X is an amino acid other than R), 328X (wherein X is an     amino acid other than Y), 395K, 459X (wherein X is an amino acid     other than S), 693 X (wherein X is an amino acid other than E), 767X     (wherein X is an amino acid other than I), 830X (wherein X is an     amino acid other than R), 897X (wherein X is an amino acid other     than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than     R), 1659X (wherein X is an amino acid other than K), 1689X (wherein     X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and     1919G. -   8. The ligand of any preceding clause, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G and/or (b) a Nav1.7 protein comprising said     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, optionally,     wherein the determining step is performed before administration of     the antibody to the human. -   9. The ligand of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G. -   10. The ligand of clause 9, wherein the assaying comprises     -   contacting the biological sample with at least one         oligonucleotide probe comprising a sequence of at least 10         contiguous nucleotides of a nucleotide sequence encoding a         Nav1.7 protein comprising said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises a         nucleotide sequence encoding said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby         forming a complex when at least one nucleotide sequence encoding         the Nav1.7 protein comprising said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is         present; and     -   detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the Nav1.7 protein comprising said amino acid selected         from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,         858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,         1464I, 1627K, 277X (wherein X is an amino acid other than R),         328X (wherein X is an amino acid other than Y), 395K, 459X         (wherein X is an amino acid other than S), 693 X (wherein X is         an amino acid other than E), 767X (wherein X is an amino acid         other than I), 830X (wherein X is an amino acid other than R),         897X (wherein X is an amino acid other than W), 1200L, 1235L,         1488X (wherein X is an amino acid other than R), 1659X (wherein         X is an amino acid other than K), 1689X (wherein X is an amino         acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. -   11. The ligand of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The ligand of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.7 protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,     optionally, wherein said human is further indicated as comprising     the nucleotide sequence of SEQ ID NO: 76, or said human is indicated     as homozygous for a nucleotide sequence encoding the Nav1.7 protein     comprising said amino acid selected from the group consisting of     136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,     1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an     amino acid other than R), 328X (wherein X is an amino acid other     than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X     (wherein X is an amino acid other than E), 767X (wherein X is an     amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. -   14. The ligand of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The ligand of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The ligand of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The ligand of any preceding clause, wherein said disease or     condition is a channelopathy or associated with a channelopathy; or     is selected from the group consisting of primary erythermalgia (PE),     paroxysmal extreme pain disorder (PEPD) and channelopathy-associated     insensitivity to pain (CIP). -   18. The ligand of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The ligand of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The ligand of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs6746030, rs3750904, rs58022607, rs4369876,     rs13402180 and rs12478318. -   21. The ligand of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is for inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation.

Methods with Gamma-4 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.7-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a huma Nav1.7 protein     that comprises an amino acid selected from the group consisting of     136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,     1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an     amino acid other than R), 328X (wherein X is an amino acid other     than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X     (wherein X is an amino acid other than E), 767X (wherein X is an     amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;     -   wherein (i) the ligand comprises a human gamma-4 heavy chain         constant region that comprises a Leu at position 189 shown in         SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73         and wherein said human comprises (i) an IGHG4*01 human heavy         chain constant region gene segment, or the human expresses         antibodies comprising human gamma-4 heavy chain constant regions         comprising a Leu at position 189 shown in SEQ ID NO: 73 or an         Arg at position 289 shown in SEQ ID NO: 73; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.7 protein comprising said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an alternative, clause 1 provides:—

A method of targeting Nav1.7 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;

wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the human is suffering from or at risk of a Nav1.7-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.7-mediated disease or condition in the human.

-   2. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of any one of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said     disease or condition is a pain disease or condition. -   3. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I and 1627K; optionally wherein said disease or     condition is a pain disease or condition. -   4. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of 277X (wherein X is an amino     acid other than R), 328X (wherein X is an amino acid other than Y),     395K, 459X (wherein X is an amino acid other than S), 693 X (wherein     X is an amino acid other than E), 767X (wherein X is an amino acid     other than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K) and 1689X (wherein X is an amino acid other     than W); optionally wherein said disease or condition is a pain     disease or condition. -   5. The method of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     and/or a Nav1.7 protein comprising said amino acid selected from the     group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,     1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X     (wherein X is an amino acid other than R), 328X (wherein X is an     amino acid other than Y), 395K, 459X (wherein X is an amino acid     other than S), 693 X (wherein X is an amino acid other than E), 767X     (wherein X is an amino acid other than I), 830X (wherein X is an     amino acid other than R), 897X (wherein X is an amino acid other     than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than     R), 1659X (wherein X is an amino acid other than K), 1689X (wherein     X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and     1919G. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes a Nav1.7 protein comprising said amino acid selected     from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,     858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,     1627K, 277X (wherein X is an amino acid other than R), 328X (wherein     X is an amino acid other than Y), 395K, 459X (wherein X is an amino     acid other than S), 693 X (wherein X is an amino acid other than E),     767X (wherein X is an amino acid other than I), 830X (wherein X is     an amino acid other than R), 897X (wherein X is an amino acid other     than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than     R), 1659X (wherein X is an amino acid other than K), 1689X (wherein     X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and     1919G and/or (b) a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G, optionally, wherein the determining step is     performed before administration of the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G. -   10. The method of clause 9, wherein the assaying comprises     contacting the biological sample with at least one oligonucleotide     probe comprising a sequence of at least 10 contiguous nucleotides of     a nucleotide sequence encoding a Nav1.7 protein comprising said     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or comprising an     antisense sequence of said contiguous nucleotides, wherein said     sequence of contiguous nucleotides comprises a nucleotide sequence     encoding said amino acid selected from the group consisting of 136I,     216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D,     1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid     other than R), 328X (wherein X is an amino acid other than Y), 395K,     459X (wherein X is an amino acid other than S), 693 X (wherein X is     an amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby forming a     complex when at least one nucleotide sequence encoding the Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     is present; and detecting the presence or absence of the complex,     wherein detecting the presence of the complex determines that the     human comprises the Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G. -   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.7 protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,     optionally, wherein said human is further indicated as comprising     the nucleotide sequence of SEQ ID NO: 76, or said human is indicated     as homozygous for a nucleotide sequence encoding the Nav1.7 protein     comprising said amino acid selected from the group consisting of     136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,     1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an     amino acid other than R), 328X (wherein X is an amino acid other     than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X     (wherein X is an amino acid other than E), 767X (wherein X is an     amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The method of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The method of any preceding clause, wherein said disease or     condition is a channelopathy or associated with a channelopathy; or     is selected from the group consisting of primary erythermalgia (PE),     paroxysmal extreme pain disorder (PEPD) and channelopathy-associated     insensitivity to pain (CIP). -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs6746030, rs3750904, rs58022607, rs4369876,     rs13402180 and rs12478318. -   21. The method of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is administered by inhaled,     intravenous or subcutaneous administration and/or is comprised in an     inhalable or injectable preparation. -   22. The method of any preceding clause, wherein the human gamma-4     heavy chain constant region of the ligand comprises the amino acid     sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof. -   23. The method of any preceding clause, wherein the human gamma-4     heavy chain constant region comprises 228P and 235E.

Ligands with Gamma-4 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of a Nav1.7-mediated disease     or condition (eg, pain) in a human in need thereof, wherein the     ligand specifically binds a huma Nav1.7 protein that comprises an     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;     -   wherein (i) the ligand comprises a human gamma-4 heavy chain         constant region that comprises a Leu at position 189 shown in         SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73         and wherein said human comprises (i) an IGHG4*01 human heavy         chain constant region gene segment, or the human expresses         antibodies comprising human gamma-4 heavy chain constant regions         comprising a Leu at position 189 shown in SEQ ID NO: 73 or an         Arg at position 289 shown in SEQ ID NO: 73; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.7 protein comprising said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting Nav1.7 in a human, wherein the ligand specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;

wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the human is suffering from or at risk of a Nav1.7-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.7-mediated disease or condition in the human.

-   2. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of any one of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said     disease or condition is a pain disease or condition. -   3. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I and 1627K; optionally wherein said disease or     condition is a pain disease or condition. -   4. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of 277X (wherein X is an amino     acid other than R), 328X (wherein X is an amino acid other than Y),     395K, 459X (wherein X is an amino acid other than S), 693 X (wherein     X is an amino acid other than E), 767X (wherein X is an amino acid     other than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K) and 1689X (wherein X is an amino acid other     than W); optionally wherein said disease or condition is a pain     disease or condition. -   5. The ligand of any preceding clause, wherein the VH gene segment     comprised by said human is a germline VH gene segment. -   6. The ligand of any preceding clause said method comprising, before     said administering, selecting a human comprising said nucleotide     sequence of (ii), wherein the human is the human of clause 1. -   7. The ligand of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     and/or a Nav1.7 protein comprising said amino acid selected from the     group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,     1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X     (wherein X is an amino acid other than R), 328X (wherein X is an     amino acid other than Y), 395K, 459X (wherein X is an amino acid     other than S), 693 X (wherein X is an amino acid other than E), 767X     (wherein X is an amino acid other than I), 830X (wherein X is an     amino acid other than R), 897X (wherein X is an amino acid other     than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than     R), 1659X (wherein X is an amino acid other than K), 1689X (wherein     X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and     1919G. -   8. The ligand of any preceding clause, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G and/or (b) a Nav1.7 protein comprising said     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, optionally,     wherein the determining step is performed before administration of     the antibody to the human. -   9. The ligand of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G. -   10. The ligand of clause 9, wherein the assaying comprises     -   contacting the biological sample with at least one         oligonucleotide probe comprising a sequence of at least 10         contiguous nucleotides of a nucleotide sequence encoding a         Nav1.7 protein comprising said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or         comprising an antisense sequence of said contiguous nucleotides,         wherein said sequence of contiguous nucleotides comprises a         nucleotide sequence encoding said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby         forming a complex when at least one nucleotide sequence encoding         the Nav1.7 protein comprising said amino acid selected from the         group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,         863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,         1627K, 277X (wherein X is an amino acid other than R), 328X         (wherein X is an amino acid other than Y), 395K, 459X (wherein X         is an amino acid other than S), 693 X (wherein X is an amino         acid other than E), 767X (wherein X is an amino acid other than         I), 830X (wherein X is an amino acid other than R), 897X         (wherein X is an amino acid other than W), 1200L, 1235L, 1488X         (wherein X is an amino acid other than R), 1659X (wherein X is         an amino acid other than K), 1689X (wherein X is an amino acid         other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is         present; and     -   detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the Nav1.7 protein comprising said amino acid selected         from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,         858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,         1464I, 1627K, 277X (wherein X is an amino acid other than R),         328X (wherein X is an amino acid other than Y), 395K, 459X         (wherein X is an amino acid other than S), 693 X (wherein X is         an amino acid other than E), 767X (wherein X is an amino acid         other than I), 830X (wherein X is an amino acid other than R),         897X (wherein X is an amino acid other than W), 1200L, 1235L,         1488X (wherein X is an amino acid other than R), 1659X (wherein         X is an amino acid other than K), 1689X (wherein X is an amino         acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. -   11. The ligand of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The ligand of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.7 protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,     optionally, wherein said human is further indicated as comprising     the nucleotide sequence of SEQ ID NO: 76, or said human is indicated     as homozygous for a nucleotide sequence encoding the Nav1.7 protein     comprising said amino acid selected from the group consisting of     136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,     1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an     amino acid other than R), 328X (wherein X is an amino acid other     than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X     (wherein X is an amino acid other than E), 767X (wherein X is an     amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. -   14. The ligand of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The ligand of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The ligand of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The ligand of any preceding clause, wherein said disease or     condition is a channelopathy or associated with a channelopathy; or     is selected from the group consisting of primary erythermalgia (PE),     paroxysmal extreme pain disorder (PEPD) and channelopathy-associated     insensitivity to pain (CIP). -   18. The ligand of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The ligand of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The ligand of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs6746030, rs3750904, rs58022607, rs4369876,     rs13402180 and rs12478318. -   21. The ligand of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is for inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation. -   22. The ligand of any preceding clause, wherein the human gamma-4     heavy chain constant region of the ligand comprises the amino acid     sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof. -   23. The ligand of any preceding clause, wherein the human gamma-4     heavy chain constant region comprises 228P and 235E.

Methods with Gamma-2 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.7-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a huma Nav1.7 protein     that comprises an amino acid selected from the group consisting of     136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,     1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an     amino acid other than R), 328X (wherein X is an amino acid other     than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X     (wherein X is an amino acid other than E), 767X (wherein X is an     amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;     -   wherein (i) the ligand comprises a human gamma-2 heavy chain         constant region that comprises an amino acid selected from the         group consisting of a Pro at position 72 shown in SEQ ID NO: 6,         an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position         76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID         NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and         wherein said human comprises (i) an IGHG2*01 human heavy chain         constant region gene segment, or the human expresses antibodies         comprising human gamma-2 heavy chain constant regions comprising         said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at         position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in         SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala         at position 257 shown in SEQ ID NO: 6; and wherein (ii) said         human comprises a nucleotide sequence encoding said Nav1.7         protein comprising said amino acid selected from the group         consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,         1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K,         277X (wherein X is an amino acid other than R), 328X (wherein X         is an amino acid other than Y), 395K, 459X (wherein X is an         amino acid other than S), 693 X (wherein X is an amino acid         other than E), 767X (wherein X is an amino acid other than I),         830X (wherein X is an amino acid other than R), 897X (wherein X         is an amino acid other than W), 1200L, 1235L, 1488X (wherein X         is an amino acid other than R), 1659X (wherein X is an amino         acid other than K), 1689X (wherein X is an amino acid other than         W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an alternative, clause 1 provides:—

A method of targeting Nav1.7 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;

wherein (i) the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the human is suffering from or at risk of a Nav1.7-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.7-mediated disease or condition in the human.

-   2. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of any one of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said     disease or condition is a pain disease or condition. -   3. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I and 1627K; optionally wherein said disease or     condition is a pain disease or condition. -   4. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of 277X (wherein X is an amino     acid other than R), 328X (wherein X is an amino acid other than Y),     395K, 459X (wherein X is an amino acid other than S), 693 X (wherein     X is an amino acid other than E), 767X (wherein X is an amino acid     other than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K) and 1689X (wherein X is an amino acid other     than W); optionally wherein said disease or condition is a pain     disease or condition. -   5. The method of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     and/or a Nav1.7 protein comprising said amino acid selected from the     group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,     1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X     (wherein X is an amino acid other than R), 328X (wherein X is an     amino acid other than Y), 395K, 459X (wherein X is an amino acid     other than S), 693 X (wherein X is an amino acid other than E), 767X     (wherein X is an amino acid other than I), 830X (wherein X is an     amino acid other than R), 897X (wherein X is an amino acid other     than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than     R), 1659X (wherein X is an amino acid other than K), 1689X (wherein     X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and     1919G. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes a Nav1.7 protein comprising said amino acid selected     from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,     858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,     1627K, 277X (wherein X is an amino acid other than R), 328X (wherein     X is an amino acid other than Y), 395K, 459X (wherein X is an amino     acid other than S), 693 X (wherein X is an amino acid other than E),     767X (wherein X is an amino acid other than I), 830X (wherein X is     an amino acid other than R), 897X (wherein X is an amino acid other     than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than     R), 1659X (wherein X is an amino acid other than K), 1689X (wherein     X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and     1919G and/or (b) a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G, optionally, wherein the determining step is     performed before administration of the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G. -   10. The method of clause 9, wherein the assaying comprises     contacting the biological sample with at least one oligonucleotide     probe comprising a sequence of at least 10 contiguous nucleotides of     a nucleotide sequence encoding a Nav1.7 protein comprising said     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or comprising an     antisense sequence of said contiguous nucleotides, wherein said     sequence of contiguous nucleotides comprises a nucleotide sequence     encoding said amino acid selected from the group consisting of 136I,     216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D,     1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid     other than R), 328X (wherein X is an amino acid other than Y), 395K,     459X (wherein X is an amino acid other than S), 693 X (wherein X is     an amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby forming a     complex when at least one nucleotide sequence encoding the Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     is present; and     -   detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the Nav1.7 protein comprising said amino acid selected         from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,         858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,         1464I, 1627K, 277X (wherein X is an amino acid other than R),         328X (wherein X is an amino acid other than Y), 395K, 459X         (wherein X is an amino acid other than S), 693 X (wherein X is         an amino acid other than E), 767X (wherein X is an amino acid         other than I), 830X (wherein X is an amino acid other than R),         897X (wherein X is an amino acid other than W), 1200L, 1235L,         1488X (wherein X is an amino acid other than R), 1659X (wherein         X is an amino acid other than K), 1689X (wherein X is an amino         acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. -   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.7 protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,     optionally, wherein said human is further indicated as comprising     the nucleotide sequence of SEQ ID NO: 76, or said human is indicated     as homozygous for a nucleotide sequence encoding the Nav1.7 protein     comprising said amino acid selected from the group consisting of     136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,     1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an     amino acid other than R), 328X (wherein X is an amino acid other     than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X     (wherein X is an amino acid other than E), 767X (wherein X is an     amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The method of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The method of any preceding clause, wherein said disease or     condition is a channelopathy or associated with a channelopathy; or     is selected from the group consisting of primary erythermalgia (PE),     paroxysmal extreme pain disorder (PEPD) and channelopathy-associated     insensitivity to pain (CIP). -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs6746030, rs3750904, rs58022607, rs4369876,     rs13402180 and rs12478318. -   21. The method of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is administered by inhaled,     intravenous or subcutaneous administration and/or is comprised in an     inhalable or injectable preparation. -   22. The method of any preceding clause, wherein the human gamma-2     heavy chain constant region of the ligand comprises IGHG2*01 amino     acid sequence or an ADCC inactivated version thereof.

Ligands with Gamma-2 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of a Nav1.7-mediated disease     or condition (eg, pain) in a human in need thereof, wherein the     ligand specifically binds a huma Nav1.7 protein that comprises an     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;     -   wherein (i) the ligand comprises a human gamma-2 heavy chain         constant region that comprises an amino acid selected from the         group consisting of a Pro at position 72 shown in SEQ ID NO: 6,         an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position         76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID         NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and         wherein said human comprises (i) an IGHG2*01 human heavy chain         constant region gene segment, or the human expresses antibodies         comprising human gamma-2 heavy chain constant regions comprising         said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at         position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in         SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala         at position 257 shown in SEQ ID NO: 6; and wherein (ii) said         human comprises a nucleotide sequence encoding said Nav1.7         protein comprising said amino acid selected from the group         consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,         1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K,         277X (wherein X is an amino acid other than R), 328X (wherein X         is an amino acid other than Y), 395K, 459X (wherein X is an         amino acid other than S), 693 X (wherein X is an amino acid         other than E), 767X (wherein X is an amino acid other than I),         830X (wherein X is an amino acid other than R), 897X (wherein X         is an amino acid other than W), 1200L, 1235L, 1488X (wherein X         is an amino acid other than R), 1659X (wherein X is an amino         acid other than K), 1689X (wherein X is an amino acid other than         W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting Nav1.7 in a human, wherein the ligand specifically binds a huma Nav1.7 protein that comprises an amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;

wherein (i) the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.7 protein comprising said amino acid selected from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X (wherein X is an amino acid other than E), 767X (wherein X is an amino acid other than I), 830X (wherein X is an amino acid other than R), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X (wherein X is an amino acid other than K), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the human is suffering from or at risk of a Nav1.7-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.7-mediated disease or condition in the human.

-   2. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of any one of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said     disease or condition is a pain disease or condition. -   3. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I and 1627K; optionally wherein said disease or     condition is a pain disease or condition. -   4. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of 277X (wherein X is an amino     acid other than R), 328X (wherein X is an amino acid other than Y),     395K, 459X (wherein X is an amino acid other than S), 693 X (wherein     X is an amino acid other than E), 767X (wherein X is an amino acid     other than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K) and 1689X (wherein X is an amino acid other     than W); optionally wherein said disease or condition is a pain     disease or condition. -   5. The ligand of any preceding clause, wherein the VH gene segment     comprised by said human is a germline VH gene segment. -   6. The ligand of any preceding clause said method comprising, before     said administering, selecting a human comprising said nucleotide     sequence of (ii), wherein the human is the human of clause 1. -   7. The ligand of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     and/or a Nav1.7 protein comprising said amino acid selected from the     group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,     1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X     (wherein X is an amino acid other than R), 328X (wherein X is an     amino acid other than Y), 395K, 459X (wherein X is an amino acid     other than S), 693 X (wherein X is an amino acid other than E), 767X     (wherein X is an amino acid other than I), 830X (wherein X is an     amino acid other than R), 897X (wherein X is an amino acid other     than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than     R), 1659X (wherein X is an amino acid other than K), 1689X (wherein     X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and     1919G. -   8. The ligand of any preceding clause, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G and/or (b) a Nav1.7 protein comprising said     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, optionally,     wherein the determining step is performed before administration of     the antibody to the human. -   9. The ligand of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G. -   10. The ligand of clause 9, wherein the assaying comprises     contacting the biological sample with at least one oligonucleotide     probe comprising a sequence of at least 10 contiguous nucleotides of     a nucleotide sequence encoding a Nav1.7 protein comprising said     amino acid selected from the group consisting of 136I, 216S, 241T,     395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,     1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other     than R), 328X (wherein X is an amino acid other than Y), 395K, 459X     (wherein X is an amino acid other than S), 693 X (wherein X is an     amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or comprising an     antisense sequence of said contiguous nucleotides, wherein said     sequence of contiguous nucleotides comprises a nucleotide sequence     encoding said amino acid selected from the group consisting of 136I,     216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D,     1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid     other than R), 328X (wherein X is an amino acid other than Y), 395K,     459X (wherein X is an amino acid other than S), 693 X (wherein X is     an amino acid other than E), 767X (wherein X is an amino acid other     than I), 830X (wherein X is an amino acid other than R), 897X     (wherein X is an amino acid other than W), 1200L, 1235L, 1488X     (wherein X is an amino acid other than R), 1659X (wherein X is an     amino acid other than K), 1689X (wherein X is an amino acid other     than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby forming a     complex when at least one nucleotide sequence encoding the Nav1.7     protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G     is present; and detecting the presence or absence of the complex,     wherein detecting the presence of the complex determines that the     human comprises the Nav1.7 protein comprising said amino acid     selected from the group consisting of 136I, 216S, 241T, 395K, 848T,     858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,     1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X     (wherein X is an amino acid other than Y), 395K, 459X (wherein X is     an amino acid other than S), 693 X (wherein X is an amino acid other     than E), 767X (wherein X is an amino acid other than I), 830X     (wherein X is an amino acid other than R), 897X (wherein X is an     amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino     acid other than R), 1659X (wherein X is an amino acid other than K),     1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,     1002L, 1161W and 1919G. -   11. The ligand of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The ligand of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.7 protein comprising said amino acid selected from the group     consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,     996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein     X is an amino acid other than R), 328X (wherein X is an amino acid     other than Y), 395K, 459X (wherein X is an amino acid other than S),     693 X (wherein X is an amino acid other than E), 767X (wherein X is     an amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,     optionally, wherein said human is further indicated as comprising     the nucleotide sequence of SEQ ID NO: 76, or said human is indicated     as homozygous for a nucleotide sequence encoding the Nav1.7 protein     comprising said amino acid selected from the group consisting of     136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,     1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an     amino acid other than R), 328X (wherein X is an amino acid other     than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X     (wherein X is an amino acid other than E), 767X (wherein X is an     amino acid other than I), 830X (wherein X is an amino acid other     than R), 897X (wherein X is an amino acid other than W), 1200L,     1235L, 1488X (wherein X is an amino acid other than R), 1659X     (wherein X is an amino acid other than K), 1689X (wherein X is an     amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. -   14. The ligand of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The ligand of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The ligand of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The ligand of any preceding clause, wherein said disease or     condition is a channelopathy or associated with a channelopathy; or     is selected from the group consisting of primary erythermalgia (PE),     paroxysmal extreme pain disorder (PEPD) and channelopathy-associated     insensitivity to pain (CIP). -   18. The ligand of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The ligand of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The ligand of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs6746030, rs3750904, rs58022607, rs4369876,     rs13402180 and rs12478318. -   21. The ligand of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is for inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation. -   22. The ligand of any preceding clause, wherein the human gamma-2     heavy chain constant region of the ligand comprises IGHG2*01 amino     acid sequence or an ADCC inactivated version thereof.

TABLE 4 1000 GENOMES PROJECT HUMAN POPULATIONS Below is a summary of the ethnic populations as per the 1000 Genomes Project sequences. Population European ancestry Utah residents (CEPH) with Northern and Western European ancestry (CEU) Toscani in Italia (TSI) British from England and Scotland (GBR) Finnish from Finland (FIN) Iberian populations in Spain (IBS) East Asian ancestry Han Chinese in Beijing, China (CHB) Japanese in Toyko, Japan (JPT) Han Chinese South (CHS) Chinese Dai in Xishuangbanna (CDX) Kinh in Ho Chi Minh City, Vietnam (KHV) Chinese in Denver, Colorado (CHD) (pilot 3 only) West African ancestry Yoruba in Ibadan, Nigeria (YRI) Luhya in Webuye, Kenya (LWK) Gambian in Western Division, The Gambia (GWD) Malawian in Blantyre, Malawi (MAB) West African Population (TBD) Americas African Ancestry in Southwest US (ASW) African American in Jackson, MS (AJM) African Caribbean in Barbados (ACB) Mexican Ancestry in Los Angeles, CA (MXL) Puerto Rican in Puerto Rico (PUR) Colombian in Medellin, Colombia (CLM) Peruvian in Lima, Peru (PEL) South Asian ancestry Ahom in the State of Assam, India Kayadtha in Calcutta, India Reddy in Hyderabad, India Maratha in Bombay, India Punjabi in Lahore, Pakistan

TABLE 5 TOIs & Related Diseases, Conditions and Example Ligands Example Disease or Human TOI Condition Example Ligand Il-17a Inflammatory Disease AIN457 Uveitis Ixekizumab Rheumatoid Arthritis Psoriasis Angiotensin II Receptor Type 1 (AT₁) Hypertension LCZ696 Neprilysin Hypertension LCZ696 Metabotropic Glutamate Receptor 5 (Mglur5) Fragile X Syndrome AFQ056 Mavoglurant A Histone Deacetylase Cancer, Eg, Multiple LBH589 Myeloma Panobinostat Diacylglycerol acyltransferase-1 (DGAT-1) Familial LCQ908 Chylomicronaemia Pradigastat Syndrome Smoothened (Smo) Basal cell carcinoma LDE225 Solid tumour Myelofibrosis Medulloblastoma Solid tumour Smoothened (Smo) receptor Basal cell carcinoma LEQ506 Solid tumors ALK Non small cell lung LDK378 carcinoma (NSCLC) phosphatidylinositol-3-kinase (PI3K) Cancer, eg, solid tumour, BKM120 mTOR breast cancer, Renal cell AKT carcinoma (RCC), Endometrial cancer, Non- small cell lung cancer (NSCLC), prostate cancer, Glioblastoma multiforme (GBM) CRPC GIST Myelofibrosis mTOR Cancer, eg, solid tumour, BEZ235 PI3K breast cancer, Renal cell AKT carcinoma (RCC), Endometrial cancer, Non- small cell lung cancer (NSCLC), prostate cancer, Glioblastoma multiforme (GBM) CRPC GIST Myelofibrosis PI3Kα Cancer, eg, solid tumour, BYL719 mTOR breast cancer, Renal cell PI3K carcinoma (RCC), AKT Endometrial cancer, Non- small cell lung cancer (NSCLC), prostate cancer, Glioblastoma multiforme (GBM) CRPC GIST Myelofibrosis Mitogen-activated ERK kinase 1 (MEK1) Cancer, eg, solid tumour, MEK162 Mitogen-activated ERK kinase 2 (MEK2) melanoma, pancreatic, colon, lung or thyroid cancer Metastasis protein kinase Acute myeloid leukemia PKC412 protein kinase C (AML) Midostaurin FLT-3 Myelodysplastic syndrome c-KIT (MDS) Aggressive systemic mastocytosis (ASM) ActRIIB Sporadic inclusion body BYM338 myositis (sIBM) bimagrumab CD19 Cancer, eg, CTL019 (formerly CART- Leukaemia, mast 19) cell leukemia, Acute Lymphoblastic Leukemia, Chronic lymphocytic leukemia (CLL) or Hematological tumors 11β-hydroxylase Cushing's syndrome LCI699 BRAF Cancer, eg, melanoma LGX818 Receptor Tyrosine Kinase Cancer, eg, solid TKI258 (formerly CHIR- FGFR tumour 258) Breast Cancer Dovitinib Endometrial cancer Hepatocellular carcinoma Renal cell carcinoma (RCC) Bladder cancer multiple myeloma (MM), hepatocellular carcinoma endometrial cancer DAC enzyme hematologic malignancy LBH589 Multiple myeloma panobinostat Myel\odysplastic syndrome (MDS) Myelofibrosis HSP90 Cancer, eg, Breast AUY922 Cancer, gastric cancer or Non- small cell lung cancer (NSCLC) FGFR Cancer, eg, solid BGJ398 tumour Breast Cancer Endometrial cancer Hepatocellular carcinoma Renal cell carcinoma (RCC) Bladder cancer multiple myeloma (MM), hepatocellular carcinoma endometrial cancer cIAP1 Cancer, eg, solid LCL161 cIAP2 tumour Breast Cancer Endometrial cancer Hepatocellular carcinoma Renal cell carcinoma (RCC) Bladder cancer multiple myeloma (MM), hepatocellular carcinoma endometrial cancer Akt Cancer, eg, Solid GDC-0068 tumour, gastric RG7440 cancer (eg, castration-resistant prostate cancer), prostate cancer, gastroesophageal junction cancer CD22 Hematologic malignancies DCDT2980S Non-Hodgkin's lymphoma RG7593 (eg, relapsed or refractory follicular non-Hodgkin's lymphoma) Diffuse large B-cell lymphoma (eg, relapsed or refractory diffuse large B- cell lymphoma) CD79b Hematologic malignancies DCDS4501A Non-Hodgkin's lymphoma RG7596 (eg, relapsed or refractory follicular non-Hodgkin's lymphoma) Diffuse large B-cell lymphoma (eg, relapsed or refractory diffuse large B- cell lymphoma) endothelin B receptor (ETBR) Melanoma, eg, metastatic DEDN6526A or unresectable melanoma RG7636 HER3 Cancer, eg, metastatic MEHD7945A epithelial tumour, RG7597 metastatic squamous cell carcinoma of the head and neck cancer, metastatic colorectal cancer EGFR Cancer, eg, metastatic MEHD7945A epithelial tumour, RG7597 metastatic squamous cell Necitumumab carcinoma of the head and neck cancer, metastatic colorectal cancer MUC16 Cancer, eg, ovarian cancer DMUC5754A (eg, platinum-resistant RG7458 ovarian cancer) sodium-dependent phosphate transport protein Cancer, eg, metastatic non- DNIB0600A 2b (NaPi2b) squamous non-small cell RG7599 lung cancer, ovarian cancer (eg, platinum-resistant ovarian cancer) PDL1 (programmed death ligand 1) Cancer, eg, solid tumour MPDL3280A metastatic melanoma RG7446 non-small cell lung cancer STEAP1 (six-transmembrane epithelial Cancer, eg, prostate cancer DSTP3086S antigen of the prostate 1) (eg, metastatic castration- RG7450 resistant prostate cancer) Bcl-2 Cancer, eg, leukaemia (eg, GDC-0199 chronic lymphocytic RG7601 leukaemia), non-Hodgkin's lymphoma Checkpoint kinase 1 (ChK1) Cancer, eg, solid tumour, GDC-0425 refractory solid tumour or RG7602 lymphoma GDC-0575 RG7741 mitogen activated protein kinase kinase Cancer, eg, solid tumour, GDC-0973 (MAPKK) melanoma (eg, metastatic RG7421 MEK melanoma) Cobimetinib GDC-0623 RG7420 epidermal growth factor domain-like-7 Cancer, eg, colorectal Parsatuzumab (EGFL7) cancer (eg, metastatic MEGF0444A colorectal cancer), NSCLC RG7414 phosphatidylinositol-3-kinase (PI3K) Cancer, eg, prostate cancer, GDC-0032 renal cell carcinoma, RG7604 endometrial cancer, breast GDC-0084 cancer (eg, HER2-negative RG7666 metastatic breast cancer, Pictilisib metastatic hormone GDC-0941 receptor-positive breast RG7321 cancer), solid tumour mTOR Cancer, eg, prostate cancer, GDC-0980 TORC1 renal cell carcinoma, RG7422 TORC2 endometrial cancer, breast PI3K cancer (eg, HER2-negative metastatic breast cancer, metastatic hormone receptor-positive breast cancer), solid tumour IL17 Autoimmune disease Secukinumab Ankylosing spondylitis; ixekizumab Asthma; Multiple myeloma; Multiple sclerosis; Polymyalgia rheumatica; Psoriasis; Psoriatic arthritis; Rheumatoid arthritis; Uveitis M1 prime segment of membrane IgE Allergic Asthma Quilizumab MEMP1972A RG7449 IFN alpha Autoimmune disease Rontalizumab Systemic lupus erythematosus PCSK9 (proprotein convertase subtilisin/kexin coronary heart disease MPSK3169A type 9) (CHD) or high risk of CHD RG7652 hyperlipidaemia hypercholesterolaemia stroke atherosclerosis a cardiovascular disease or condition a condition associated with elevated LDL Vascular Endothelial Growth Factor-A Diabetic Macular Edema EYLEA ® (VEGF-A) (DME), Aflibercept Placental Growth Factor (PlGF) Branch Retinal Vein AVASTIN ™ Occlusion (BRVO) LUCENTIS ™ Wet age-related macular degeneration (Wet AMD) IL-6 receptor Inflammatory disease, eg, Sarilumab rheumatoid arthritis REGN88 Uveitis (eg, non-infectious uveitis) Ankylosing spondylitis; Cancer PCSK9 (proprotein convertase subtilisin/kexin coronary heart disease Alirocumab type 9) (CHD) or high risk of CHD REGN727 hyperlipidaemia LY3015014 hypercholesterolaemia stroke atherosclerosis a cardiovascular disease or condition a condition associated with elevated LDL NGF Osteoarthritis Fasinumab Pain REGN475 IL-4 receptor alpha Allergic asthma Dupilumab eosinophilic asthma REGN668 IL-13 receptor Atopic dermatitis delta-like ligand-4 (Dll4) Cancer Enoticumab REGN421 angiopoietin-2 (Ang2) Cancer Nesvacumab REGN910 GDF8 Metabolic disorder REGN1033 cancer, obesity, diabetes, LY2495655 arthritis, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolic syndromes (including, but not limited to diabetes, obesity, nutritional disorders, organ atrophy, chronic obstructive pulmonary disease and anorexia) Disuse muscle atrophy cancer-related cachexia ERBB3 Cancer REGN1400 angiopoietin-1 (Ang1) Cancer, eg, ovarian cancer AMG386 angiopoietin-1 (Ang2) VEGF receptor Cancer Motesanib PDGF receptor NSCLC AMG 706 Stem cell factor receptor Breast cancer Thyroid cancer type 1 insulin-like growth factor receptor Cancer Ganitumab (IGF1R) Breast cancer Linsitinib Pancreatic cancer ASP7487 hepatocyte growth factor (HGF) Cancer Rilotumumab Breast cancer Pancreatic cancer HER3 Cancer AMG 888 ErbB3 Breast cancer U3-1287 Pancreatic cancer IL-17 receptor Inflammatory disease AMG 827 Asthma brodalumab Psoriasis sclerostin Bone-related condition AMG 785 postmenopausal CDP7851 osteoporosis fracture healing glucokinase Diabetes AMG 151 ARRY-403 PCSK9 (proprotein convertase subtilisin/kexin coronary heart disease AMG 145 type 9) (CHD) or high risk of CHD Evolocumab hyperlipidaemia hypercholesterolaemia stroke atherosclerosis a cardiovascular disease or condition a condition associated with elevated LDL VLA 2 Inflammatory bowel SAR339658 Integrin α2β1 disease IL-4 Idiopathic pulmonary SAR156597 IL-13 fibrosis lysophosphatidic acid receptor Systemic sclerosis SAR100842 LPA-1 fibrosis LPA-3 Androgen receptor Cancer, eg, prostate cancer, MDV3100 breast cancer enzalutamide HER1 Cancer, eg, NSCLC Erlotinib EGFR ERBITUX ™ VECTIBIX ™ VEGF receptor 1 Cancer, eg, colorectal ASP4130 VEGF receptor 2 cancer, breast cancer tivozanib VEGF receptor 3 JAK Inflammatory disease, eg, ASP015K JAK1 rheumatoid arthritis Baricitinib JAK2 Diabetic nephropathy CD40 Prevention of organ ASP1240 transplant rejection GnRH Endometriosis ASP1707 Cancer, eg, prostate cancer degarelix PDE9 Lower unirary tract ASP4901 symptoms associated with benign prostatic hyperplasia TNF alpha Inflammatory disease, eg, Certolizumab pegol rheumatoid arthritis, psoriasis, chrohn's disease, IBD Programmed cell death protein 1 Cancer Nivolumab Chronic myelocytic MK-3475 leukemia; Hepatitis C virus infection; Hepatocellular carcinoma; Hodgkins disease; Melanoma; Multiple myeloma; Non- Hodgkin lymphoma; Non- small-cell lung cancer; Renal cell carcinoma; Solid tumor; Stage IV melanoma Hepatocyte growth factor Cancer onartuzumab MET Glioblastoma; Hepatocellular carcinoma; Metastatic colorectal cancer; Metastatic non small cell lung cancer; Metastatic stomach cancer; Non-small-cell lung cancer Angiopoietin ligand-1 Breast tumor; Cancer; trebananib Angiopoietin ligand-2 Colorectal tumor; Fallopian Tek tyrosine kinase receptor tube cancer; Gastrointestinal tumor; Glioblastoma; Hepatocellular carcinoma; Metastatic esophageal cancer; Metastatic gastrointestinal cancer; Metastatic non small cell lung cancer; Metastatic ovary cancer; Metastatic renal cancer; Ovary tumor; Peritoneal tumor; Transitional cell carcinoma CD37 modulator Cancer elotuzumab Lymphocyte function antigen-3 receptor Multiple myeloma SLAM family member 7 IL-2 Multiple sclerosis daclizumab IL-2 receptor alpha EGFR Cancer necitumumab Metastatic non small cell lung cancer; Solid tumor IL-5 Asthma; Eosinophilic reslizumab esophagitis B-lymphocyte cell adhesion molecule Cancer, eg Acute Inotuzumab CD22 lymphoblastic leukemia; inotuzumab ozogamicin Follicle center lymphoma; epratuzumab Non-Hodgkin lymphoma; moxetumomab Systemic lupus moxetumomab pasudotox erythematosus, hairy cell leukaemia IL1 beta Acne vulgaris; gevokizumab Atherosclerosis; Behcets disease; Cardiovascular disease; Dermatomyositis; Insulin dependent diabetes; Multiple myeloma; Osteoarthritis; Paraproteinemia; Polymyositis; Pyoderma gangrenosum; Scleritis; Uveitis CD20 Cancer Ocrelizumab Multiple sclerosis ofatumumab follicular lymphoma (eg, refractory or relapsed) diffuse large B cell lymphoma (eg, relapsed) chronic lymphocytic leukaemia (eg, first line therapy or relapsed) IL-23 Crohns disease; tildrakizumab Inflammatory disease; Psoriasis BAFF Autoimmune disease Belimumab Neutrokine alpha systemic lupus Benlysta ™ erythematosus Tabalumab Multiple myeloma vasculitis IL5 Asthma mepolizumab IL6 Inflammatory disease sirukumab rheumatoid arthritis Lp-PLA2 Atherosclerosis darapladib diabetic macular oedema CCR9 chemokine receptor Inflammatory disease Vercirnon rheumatoid arthritis Crohn's disease DOPA decarboxylase Parkinson's Disease Patrome Her2 Cancer, eg, gastric cancer, Tyverb ™ EGFR breast cancer, head and Tykerb ™ neck squamous cell cancer, lapatinib ADP receptor Cardiovascular disease or Brilinta condition Brilique Thrombosis, eg, arterial thrombosis VEGFR Cancer, eg, Caprelsa EGFR medullary thyroid cancer LABA Respiratory disease or PT003 GFF LAMA condition, eg, COPD Factor Xa thromboembolism apixaban OX40 ligand Asthma Oxelumab Graft-versus-host disease CTLA-4 Cancer, eg, melanoma Tremelimumab CD152 Autoimmune disease Ticilimumab ipilimumab PD-L1 Cancer, eg, solid tumour, MPDL3280A kidney cancer, lung cancer, MEDI4736 melanoma, NSCLC, multiple myeloma Autoimmune disease PD-1 Cancer, eg, solid tumour, Nivolumab kidney cancer, lung cancer, Keytruda melanoma, NSCLC, AMP-514 multiple myeloma AMP-224 Autoimmune disease LIGHT TNFSF14 CD40 ligand JAK Inflammatory disease, eg, tofacitinib rheumatoid arthritis, psoriasis, chrohn's disease, IBD, ulcerative colitis, Psoriatic Arthritis Nerve Growth Factor Pain tanezumab Osteoarthritis pain Her1 receptor Cancer, eg, Non-Small Cell dacomitinib Her2 receptor Lung Cancer Her4 receptor c-MET Cancer, eg, Non-Small Cell crizotinib ALK Lung Cancer Programmed cell death 1 receptor Cancer, eg, renal cell Nivolumab carcinoma SLAMF7 Cancer, eg, multiple Elotuzumab CD319 myeloma CD30 Cancer, eg, Hodgkin Brentuximab lymphoma, systemic Brentuximab vedotin anaplastic large cell lymphoma, T-cell lymphoma GPR40 Diabetes, eg, diabetes Fasiglifam DPP-4 mellitus trelagliptin VEFGR-1 receptor Cancer, eg, non-squamous Motesanib VEFGR-2 receptor non-small cell lung cancer Motesanib diphosphate VEFGR-3 receptor PDGFR cKit amyloid β Alzheimer's disease Solanezumab TNF alpha Inflammatory disease, eg, SIMPONI ™ rheumatoid arthritis, HUMIRA ™ psoriasis, chrohn's disease, REMICADE ™ IBD, ulcerative colitis, ENBREL ™ Psoriatic Arthritis Adalimumab IL-21 Autoimmune disease or Agonist or antagonist condition antibody specific for Inflammatory disease or human IL-21 condition NNC0114-0005 Rheumatoid arthritis NNC0114-0006 Crohn's disease NN8828 IBD ATR-107 Ulcerative colitis Said or an anti-IL-21 Systemic lupus antibody in combination erythematosus (SLE) with an agent selected from Graft versus host disease the group consisting of Cancer ipilimumab (eg, to treat Metastatic melanoma melanoma), an anti-PD1 Renal cell carcinoma antibody (eg, to treat solid Melanoma tumours), sunitinib (eg, to Solid tumours treat renal cell carcinoma), Acute myeloid leukaemia rituximab (eg, to treat Non- Non-Hodgkin's lymphoma Hodgkin's lymphoma), Ovarian cancer sorafenib (eg, to treat renal Colorectal cancer cell carcinoma), doxorubicin (eg, to treat ovarian cancer) and cetuximab (eg, to treat colorectal cancer). TIGIT immuno-oncology condition DMAM-1 (immuno-oncology) CD96 (immuno-oncology) DR3 (agonist for immuno-oncology) TL1A, DR3 (blocker for inflammatory disease) DMAM-1 immuno-oncology condition CD96 immuno-oncology condition DR3 immuno-oncology condition Agonist inflammatory disease Antagonist TL1A inflammatory disease TIM3 CD200 CSF1R LAG3 GITR CD40

TABLE 6 PCSK9 SEQUENCES FORM/ SEQ ALLELE VERSION SEQUENCE ID NO: AMINO ACID SEQUENCES Italics = signal sequence 1-30 Courier = pro peptide 31-152 lower case = catalytic domain 153-449 UPPER CASE = C-terminal domain 450-692 Underlined = residues changed from allele a in other sequences (aa residue number shown) a Pro-Form                                             46     53  1 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvineawfpedqrvltpnlva 443                           474 alppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ a Pro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER  2 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMAT 474 AIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSEEAVTAVAICCRSRHLAQASQELQ a Mature form sipwnieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbf  3 hrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqp vgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaq dqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepe                   425               443 itlaelrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPT     474 RMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQ ANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREA                       619 620 SIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVR            670 SRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ f Pro-Form                                             46     53  4 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvineawfpedqrvltpnlva 443                           474 alppsthGAGWQLFCRTVWSAHSGPTRMATA V ARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ f Pro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER  5 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMAT 474 A V ARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSEEAVTAVAICCRSRHLAQASQELQ f, p, aj Mature form sipwnieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbf  6 hrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqp vgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaq dqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepe                   425               443 itlaelrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPT     474 RMATA V ARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQ ANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREA                       619 620 SIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVR            670 SRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ c Pro-Form                                             46     53  7 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvineawfpedqrvltpnlva 443                           474 alppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ c Pro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER  8 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMAT 474 AIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSE G AVTAVAICCRSRHLAQASQELQ c, q Mature form sipwnieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbf  9 hrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqp vgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaq dqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepe                   425               443 itlaelrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPT     474 RMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQ ANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREA                       619 620 SIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVR            670 SRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ r Pro-Form                                             46     53 10 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvineawfpedqrvltpnlva 443                           474 alppsthGAGWQLFCRTVWSAHSGPTRMATA V ARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ r Pro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER 11 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMAT 474 A V ARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSE G AVTAVAICCRSRHLAQASQELQ r Mature form sipwnieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbf 12 hrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqp vgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaq dqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepe                   425               443 itlaelrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPT     474 RMATA V ARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQ ANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREA                       619 620 SIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVR            670 SRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ p Pro-Form                                             46     53 13 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGL V EAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvineawfpedqrvltpnlva 443                           474 alppsthGAGWQLFCRTVWSAHSGPTRMATA V ARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ p Pro-Form QEDEDGDYEELVLALRSEEDGL V EAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER 14 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMAT 474 A V ARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSEEAVTAVAICCRSRHLAQASQELQ m Pro-Form                                             46     53 15 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvineawfpedqrvltpnlva 443                           474 t lppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ m Pro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER 16 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvineawfpedqrvltpnlva t lppsthGAGWQLFCRTVWSAHSGPTRMAT 474 AIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSEEAVTAVAICCRSRHLAQASQELQ m Mature form sipwnieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbf 17 hrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqp vgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaq dqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepe                   425               443 itlaelrqrlihfsakdvineawfpedqrvltpnlva t lppsthGAGWQLFCRTVWSAHSGPT     474 RMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQ ANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREA                       619 620 SIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVR            670 SRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ e Pro-Form                                             46     53 18 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvi s eawfpedqrvltpnlva 443                           474 alppsthGAGWQLFCRTVWSAHSGPTRMATA V ARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ e Pro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER 19 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvi s eawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMAT 474 A V ARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSEEAVTAVAICCRSRHLAQASQELQ e Mature form sipwnieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbf 20 hrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqp vgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaq dqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepe                   425               443 itlaelrqrlihfsakdvi s eawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPT     474 RMATA V ARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQ ANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREA                       619 620 SIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVR            670 SRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ h Pro-Form                                             46     53 21 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvineawfpedqrvltpnlva 443                           474 t lppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAP PEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ h Pro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER 22 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvineawfpedqrvltpnlva t lppsthGAGWQLFCRTVWSAHSGPTRMAT 474 AIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAP PEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSEEAVTAVAICCRSRHLAQASQELQ h Mature form sipwnieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbf 23 hrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqp vgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaq dqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepe                   425               443 itlaelrqrlihfsakdvineawfpedqrvltpnlva t lppsthGAGWQLFCRTVWSAHSGPT     474 RMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQ ANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREA                       619 620 SIHASCCHAPGLECKVKEHGIPAP PEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVR            670 SRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ aj Pro-Form                                             46     53 24 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLAL L SEEDGLAEAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvineawfpedqrvltpnlva 443                           474 alppsthGAGWQLFCRTVWSAHSGPTRMATA V ARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ aj Pro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER 25 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMAT 474 AIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSEEAVTAVAICCRSRHLAQASQELQ q Pro-Form                                             46     53 26 with Signal MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLAL L SEEDGLAEAPEHGTTAT Sequence FHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKM SGDLLELALKLPHVDYIEEDSSVFAQsipwnieritppryradeygppdggslvevyiidtsi qsdhrelegrvmvtdfenvpeedgbfhrqaskcdshgthlagvvsgrdagvakgasmrslrvl ncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaag nfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgqdiigassdcstcf                                             425 vsqsgtsqaaahvagiaammisaepeitlaelrqrlihfsakdvineawfpedqrvltpnlva 443                           474 alppsthGAGWQLFCRTVWSAHSGPTRMATA V ARCAPDEELLSCSSFSRSGKRRGERMEAQGG KLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEV                                                 619 620 EDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWT                                      670 LTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ q Pro-Form QEDEDGDYEELVLALRSEEDGL V EAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSER 27 TARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQsipw nieritppryradeygppdggslvevyiidtsiqsdhrelegrvmvtdfenvpeedgbfhrqa skcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgpl vvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpv tlgtlgtnfgrcvdlfapgqdiigassdcstcfvsqsgtsqaaahvagiaammisaepeitla               425               443 elrqrlihfsakdvineawfpedqrvltpnlvaalppsthGAGWQLFCRTVWSAHSGPTRMAT 474 AIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCS VHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHA                   619 620 SCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDV        670 STTGSTSE G AVTAVAICCRSRHLAQASQELQ NUCLEOTIDE SEQUENCES italics = nucleotide sequence encoding signal sequence (nucleotides 1-90) Courier = nucleotide sequence encoding pro peptide (nucleotides 91-456) lower case = nucleotide sequence encoding catalytic domain (nucleotides 457-1346) UPPER CASE = nucleotide sequence encoding C-terminal domain (nucleotides 1347-2076) a ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 28 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGAGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC f ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 29 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCC GTCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGAGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC c ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 30 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGGGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC r ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 31 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCC GTCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGGGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC p ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 32 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCGTTCCGAGGAGGACGGCCTGGTCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCC GTCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGAGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC m ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 33 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggcc accctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGAGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC e ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 34 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcagtgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCC GTCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGAGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC h ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 35 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggcc accctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCCGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGAGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC aj ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 36 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCTTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCC GTCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGAGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC q ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTG 37 CTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTG    R46L CGT to CTT      A53V GCC to GTC CTAGCCTTGCGTTCCGAGGAGGACGGCCTGGTCGAAGCACCCGAGCACGGAACCACAGCCACC TTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAG GAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGG GGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATG AGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCC TCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcg gatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcata cagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgag gaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggca ggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtg ctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaa agccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagc cgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgcc ggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagtt ggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgc tgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgc tttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatg ctgtctgccgagccggagctcaccctggccgagttgaggcagagactgatccacttctctgcc      N425S AAT to AGT aaagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgacccccaaccttggt A443T GCC to ACC ggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTC                            I474V ATC to GTC AGCACACTCGGGGCCTACACGGATGGCCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCT GCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGG GGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAG GTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCAT GGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGA GGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTG CGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAA                Q519P CAG to CCG E620G GAG to GGG AGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTG GACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGA                                                E670G GAG to GGG CAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGGGGCCGT GACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTG AC

TABLE 7 Human VH3-23 Variant Alleles VH3-23 Cumulative allele haplotype frequency SNPs a (=VH3-23*04) 0.0983 rs56069819 d 0.0087 rs56069819 rs61750837 rs61752504 e 0.0046 rs56069819 rs1064090 rs1055799 j 0.0009 rs56069819 rs1055799 u 0.0005 rs56069819 rs1064091 s 0.0005 rs56069819 rs1064091 rs61752504 rs61750837 r 0.0005 rs56069819 rs1064090 TOTAL: 0.114

TABLE 8 Exemplary anti-PCSK9 antibodies and/or antibody fragments SEQ ID NOs comprising an anti-PCSK9 Patent or patent monoclonal antibody or fragment thereof Pblication Light chain complementary determining regions US20120020975 A1 (CDRL) SEQ ID NO: 5, 7, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 270, 271, 272, 273, 275, 277, 286, 287, 288, 297, 299, 301, 405, 407, 409, 411, 413, 415, 417, 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485; Heavy chain complementary determining regions (CDRH) SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 278, 289, 290, 291, 292, 298, 300, 302, 401, 404, 406, 408, 410, 412, 414, 416, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20120027765A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 465; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 463; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, U.S. Pat. No. 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 8,168,762B2 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 465; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 463; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20120020976A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 222, 229, 238, 405, 407, 409, 411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 247, 256, 265, 404, 406, 408, 410, 412, 414, 416; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130085265A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130079501A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20120213797A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 158, 162, 395, 473, 477; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 180, 175, 308, 368, 471, 475; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20120251544A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130052201A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130058944A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130079502A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130245235A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416;

TABLE 9 Human variable & constant variants distributed over several human ethnic populations - useful for ligand tailoring Nucleotide Hom Human Variation² Freq⁷ Gene Example Amino Acid (NCBI dbSNP Variant (Het + Segment Human Position & reference Nucleotide Human No. Het Hom Cum Type Allele¹ Variation number)³ Position Populations⁴ Individs⁵ Freq⁶ freq⁸) Freq⁹ IGHG1 IGHG1*01 204D GAT 14: 106208086 A 153 0.400 0.096 0.296 (CH3 (forward strand) (European (0.496) variation) ancestry) IGHG1*03 204E GAG 14: 106208086 A 366 0.400 0.504 0.704 (CH3 (rs1045853) (forward strand) (European (0.904) variation) ancestry IGHG1*01 206L CTG 14: 106208082 A 0.358 0.104 0.283 (CH3 (forward strand) (European (0.462) variation) ancestry) IGHG1*03 206M ATG 14: 106208082 A 0.358 0.538 0.717 (CH3 (rs11621259) (forward strand) (European (0.896) variation) ancestry) IGHG2 IGHG2*01 72P CCC 14: 106110914 B 0.336 0.540 0.708 (CH1 (forward strand) (0.876) variation) IGHG2*02 72T ACC 14: 106110914 B 0.336 0.124 0.292 (CH1 (rs11627594) (forward strand) variation) IGHG2*01 75N AAC 14: 106110904 A 0.007 0.993 0.997 (CH1 (forward strand) variation) IGHG2*04 75S AGC 14: 106110904 A 0.007 0.004 (CH1 (rs201590297) (forward strand) variation) IGHG2*01 76F TTC (CH1 variation) IGHG2*04 76L TTG (CH1 variation) IGHG2*01 161V GTG 14: 10611013 B 0.342 0.539 0.711 (CH2 (forward strand) (0.881) variation) IGHG2*02 161M ATG 14: 10611013 B 0.342 0.118 0.289 (CH2 (rs8009156) (forward strand) (0.46) variation) IGHG2*01 257A GCC 14: 106109752 C 0.199 0.493 0.592 (CH3 (forward strand) (0.692) variation) D 0.007 0.992 0.995 (0.999) IGHG2*06 257S TCC 14: 106109752 C 0.199 0.308 0.408 (CH3 (rs4983499) (forward strand) (0.507) variation) D 0.007 0.002 0.005 (0.009) IGHG4*01 189L CTG 14: 105624992 B 1047 0.315 0.644 0.801 IGHG4 (CH2 (forward strand) (0.959) variation) IGHG4*02 189V GTG 14: 105624992 B 389 0.315 0.041 0.199 (CH2 (rs8015545) (forward strand) (0.356) variation) IGHG4*01 289R AGG 14: 105624594 (CH3 (forward strand) variation) IGHG4*03 289K AAG 14: 105624594 (CH3 (rs77498506) (forward strand) variation) Table Footnotes: ¹IMGT notation (ww.imgt.org); refer to figures for other alleles comprising this variation. ²SNP Underlined in Codon. ³NCBI dbSNP Build 138 released on Apr. 25, 2013. ⁴Human population used for representative variant frequency analysis. Populations A This population included 662 participants of European descent from the ClinSeq project, all of whom had undergone whole-exome sequencing using Agilent's 38 Mb or 50 Mb capture kit. B 1000 Genomes database. C ESP6500: African_American. D ESP6500: European_American. ⁵Number of individuals in representative population found to have the allele. ⁶Heterozygous human genotype frequency, ie, cumulative frequency of all genotypes having one occurrence of the variant allele and one occurrence of another allele (heterozygous state), eg, ac genotype in the population. ⁷Homozygous human genotype frequency, ie, cumulative frequency of two occurrences of the variant allele (homozygous state), eg, cc genotype in the population. ⁸Total human genotype frequency, ie, total of heterozygous plus homozygous human genotype frequencies. ⁹Cumulative human allele frequency of all occurrences of the variant allele in the population.

TABLE 10 FURTHER SEQUENCES SEQ ID Human NO: Allele Nucleotide/Amino Acid Sequence HEAVY CHAIN ALLELES 41 IGHG1*01 gcctccaccaagggcccatcggtcttccccctggcaccctcctccaaga (CH1 + Hinge + gcacctctggg CH2 + CH3 + CH-S) ggcacagcggccctgggctgcctggtcaaggactacttccccgaaccgg tgacggtgtcg tggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcc tacagtcctca ggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgg gcacccagacc tacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaaga aagttgagccc aaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaac tcctgggggga ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct cccggacccct gaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtca agttcaactgg tacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagg agcagtacaac agcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggc tgaatggcaag gagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgaga aaaccatctcc aaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccat cccgggatgag ctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatc ccagcgacatc gccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtg ctggactccgacggctccttcttcctctacagcaagctcaccgtggaca agagcaggtgg cagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcaca accactacacg cagaagagcctctccctgtctccgggtaaa 42 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSR D E L TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK D  = position 204 L  = position 206 43 IGHG2*01 gcctccaccaagggcccatcggtcttccccctggcgccctgctccagga (CH1 + Hinge + gcacctccgag CH2 + CH3 + CH-S) agcacagccgccctgggctgcctggtcaaggactacttccccgaaccgg tgacggtgtcg tggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcc tacagtcctca ggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcg gcacccagacc tacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaaga cagttgagcgc aaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggac cgtcagtcttc ctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg aggtcacgtgc gtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggt acgtggacggc gtggaggtgcataatgccaagacaaagccacgggaggagcagttcaaca gcacgttccgt gtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaagg agtacaagtgc aaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctcca aaaccaaaggg cagccccgagaaccacaggtgtacaccctgcccccatcccgggaggaga tgaccaagaac caggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcg ccgtggagtgg gagagcaatgggcagccggagaacaactacaagaccacacctcccatgc tggactccgac ggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggc agcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgc agaagagcctc tccctgtctccgggtaaa 44 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSS GLYSLSSVVTV P SS NF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG V EVHNAKTK PREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYT LPPSREEMTKN QVSLTCLVKGFYPSDI A VEWESNGQPENNYKTTPPMLDSDGSFFLYSKL TVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK P  = position 72 N  = position 75 F  = position 76 V  = position 161 A  = position 257 45 IGHV1-J8*01 caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcct cagtgaaggtc tcctgcaaggcttctggttacacctttaccagctatggtatcagctggg tgcgacaggcc cctggacaagggcttgagtggatgggatggatcagcgcttacaatggta acacaaactat gcacagaagctccagggcagagtcaccatgaccacagacacatccacga gcacagcctac atggagctgaggagcctgagatctgacgacacggccgtgtattactgtg cgagaga 46 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAP GQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCAR 47 IGHV1-46*01 caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct cagtgaaggtt tcctgcaaggcatctggatacaccttcaccagctactatatgcactggg tgcgacaggcc cctggacaagggcttgagtggatgggaataatcaaccctagtggtggta gcacaagctac gcacagaagttccagggcagagtcaccatgaccagggacacgtccacga gcacagtctac atggagctgagcagcctgagatctgaggacacggccgtgtattactgtg cgagaga 48 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAP GQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED TAVYYCAR LIGHT CHAIN ALLELES 49 IGKC*01 cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc agttgaaatct ggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg ccaaagtacag tggaaggtggataacgccctccaatcgggtaactcccaggagagtgtca cagagcaggac agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaag cagactacgag aaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgc ccgtcacaaag agcttcaacaggggagagtgt 50 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHK V YA C EVTHQGLSSPVTKSFNRGEC V  = position 84 C  = position 87 51 IGLC2*01 ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctg aggagcttcaa gccaacaaggccacactggtgtgtctcataagtgacttctacccgggag ccgtgacagtg gcttggaaagcagatagcagccccgtcaaggcgggagtggagaccacca caccctccaaa caaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctg agcagtggaag tcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtgg agaagacagtg gcccctacagaatgttca 52 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV KAGVETTTPSK QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 53 IGKV4-1*01 atggtgttgcagacccaggtcttcatttctctgttgctctggatctctg gtgcctacggg gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcg agagggccacc atcaactgcaagtccagccagagtgttttatacagctccaacaataaga actacttagct tggtaccagcagaaaccaggacagcctcctaagctgctcatttactggg catctacccgg gaatccggggtccctgaccgattcagtggcagcgggtctgggacagatt tcactctcacc atcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaat attatagtact cctcc 54 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLA WYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQYYST P 55 IGKV1-J3*02 atggacatgagggtccccgctcagctcctggggcttctgctgctctggc tcccagcaggt gccagatgtgccatccagttgacccagtctccatcctccctgtctgcat ctgtaggagac agagtcaccatcacttgccgggcaagtcagggcattagcagtgctttag cctggtatcag cagaaaccagggaaagctcctaagctcctgatctatgatgcctccagtt tggaaagtggg gtcccatcaaggttcagcggcagtggatctgggacagatttcactctca ccatcagcagc ctgcagcctgaagattttgcaacttattactgtcaacagtttaatagtt accctcagtgc cagatgtgccatccagttgacccagtctccatcctccctgtctgcatct gtaggagacag agtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcc tggtatcagca gaaaccagggaaagctcctaagctcctgatctatgatgcctccagtttg gaaagtggggt cccatcaaggttcagcggcagtggatctgggacagatttcactctcacc atcagcagcct gcagcctgaagattttgcaacttattactgtcaacagtttaatagttac cctca 57 IGKJ2*01 tgtacacttttggccaggggaccaagctggagatcaaac 58 YTFGQGTKLEIK 59 IGLJ2*01 tgtggtattcggcggagggaccaagctgaccgtcctag 60 VVFGGGTKLTVL 61 An IGHG1*01 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG Heavy Chain VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV Constant EPKSCDKTHTCPPCPAPELLGGPS Region VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 62 An IGKC*01 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS Kappa Light GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV Chain TKSFNRGEC Constant Region 63 An IGHG2*01 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Heavy Chain VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV Constant ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH Region EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 64 An IGLC2*01 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK Lambda AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT Light Chain VAPTECS Constant Region 65 An IGHG2*01 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Heavy Chain VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV Constant ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH Region EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK EYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 66 An IGKC*01 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS Kappa Light GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV Chain TKSFNRGEC Constant Region HUMAN IL4Ra SEQUENCES 67 Amino Acid MGWLCSGLLFPVSCLVLLQVASSGNMKVLQEPTCVSDYMSISTCEWKMNGPTNC Sequence STELRLLYQLVFLLSEAHTC I PENNGGAGCVCHLLMDDVVSADNYTLDLWAGQQ LLWKGSFKPSEHVKPRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLYNHLTYAVN IWSENDPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRAWAQCYNTTWSEWS PSTKWHNSYREPFEQHLLLGVSVSCIVILAVCLLCYVSITKIKKEWWDQIPNPA RSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLEHNMKRDEDP HKAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVE EEKGSFCASPESSRDDFQEGR E GIVARLTESLFLDLLGEENGGFCQQDMGES C L LPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTE TPLVIAGNPAYRSFSN S LSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPT TVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGY Q EFVHAVEQGGTQASAVVG LGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPV PVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQATDP LVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDR S SPPT TPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPK IVNFVSVGPTYMRVS I  = position 75 E  = position 400 C  = position 431 S  = position 503 Q  = position 576 S  = position 752 68 Nucleotide ATGGGGTGGCTTTGCTCTGGGCTCCTGTTCCCTGTGAGCTGCCTGGTCCTGCTG Sequence CAGGTGGCAAGCTCTGGGAACATGAAGGTCTTGCAGGAGCCCACCTGCGTCTCC GACTACATGAGCATCTCTACTTGCGAGTGGAAGATGAATGGTCCCACCAATTGC AGCACCGAGCTCCGCCTGTTGTACCAGCTGGTTTTTCTGCTCTCCGAAGCCCAC ACGTGTATCCCTGAGAACAACGGAGGCGCGGGGTGCGTGTGCCACCTGCTCATG GATGACGTGGTCAGTGCGGATAACTATACACTGGACCTGTGGGCTGGGCAGCAG CTGCTGTGGAAGGGCTCCTTCAAGCCCAGCGAGCATGTGAAACCCAGGGCCCCA GGAAACCTGACAGTTCACACCAATGTCTCCGACACTCTGCTGCTGACCTGGAGC AACCCGTATCCCCCTGACAATTACCTGTATAATCATCTCACCTATGCAGTCAAC ATTTGGAGTGAAAACGACCCGGCAGATTTCAGAATCTATAACGTGACCTACCTA GAACCCTCCCTCCGCATCGCAGCCAGCACCCTGAAGTCTGGGATTTCCTACAGG GCACGGGTGAGGGCCTGGGCTCAGTGCTATAACACCACCTGGAGTGAGTGGAGC CCCAGCACCAAGTGGCACAACTCCTACAGGGAGCCCTTCGAGCAGCACCTCCTG CTGGGCGTCAGCGTTTCCTGCATTGTCATCCTGGCCGTCTGCCTGTTGTGCTAT GTCAGCATCACCAAGATTAAGAAAGAATGGTGGGATCAGATTCCCAACCCAGCC CGCAGCCGCCTCGTGGCTATAATAATCCAGGATGCTCAGGGGTCACAGTGGGAG AAGCGGTCCCGAGGCCAGGAACCAGCCAAGTGCCCACACTGGAAGAATTGTCTT ACCAAGCTCTTGCCCTGTTTTCTGGAGCACAACATGAAAAGGGATGAAGATCCT CACAAGGCTGCCAAAGAGATGCCTTTCCAGGGCTCTGGAAAATCAGCATGGTGC CCAGTGGAGATCAGCAAGACAGTCCTCTGGCCAGAGAGCATCAGCGTGGTGCGA TGTGTGGAGTTGTTTGAGGCCCCGGTGGAGTGTGAGGAGGAGGAGGAGGTAGAG GAAGAAAAAGGGAGCTTCTGTGCATCGCCTGAGAGCAGCAGGGATGACTTCCAG GAGGGAAGGGAGGGCATTGTGGCCCGGCTAACAGAGAGCCTGTTCCTGGACCTG CTCGGAGAGGAGAATGGGGGCTTTTGCCAGCAGGACATGGGGGAGTCATGCCTT CTTCCACCTTCGGGAAGTACGAGTGCTCACATGCCCTGGGATGAGTTCCCAAGT GCAGGGCCCAAGGAGGCACCTCCCTGGGGCAAGGAGCAGCCTCTCCACCTGGAG CCAAGTCCTCCTGCCAGCCCGACCCAGAGTCCAGACAACCTGACTTGCACAGAG ACGCCCCTCGTCATCGCAGGCAACCCTGCTTACCGCAGCTTCAGCAACTCCCTG AGCCAGTCACCGTGTCCCAGAGAGCTGGGTCCAGACCCACTGCTGGCCAGACAC CTGGAGGAAGTAGAACCCGAGATGCCCTGTGTCCCCCAGCTCTCTGAGCCAACC ACTGTGCCCCAACCTGAGCCAGAAACCTGGGAGCAGATCCTCCGCCGAAATGTC CTCCAGCATGGGGCAGCTGCAGCCCCCGTCTCGGCCCCCACCAGTGGCTATCAG GAGTTTGTACATGCGGTGGAGCAGGGTGGCACCCAGGCCAGTGCGGTGGTGGGC TTGGGTCCCCCAGGAGAGGCTGGTTACAAGGCCTTCTCAAGCCTGCTTGCCAGC AGTGCTGTGTCCCCAGAGAAATGTGGGTTTGGGGCTAGCAGTGGGGAAGAGGGG TATAAGCCTTTCCAAGACCTCATTCCTGGCTGCCCTGGGGACCCTGCCCCAGTC CCTGTCCCCTTGTTCACCTTTGGACTGGACAGGGAGCCACCTCGCAGTCCGCAG AGCTCACATCTCCCAAGCAGCTCCCCAGAGCACCTGGGTCTGGAGCCGGGGGAA AAGGTAGAGGACATGCCAAAGCCCCCACTTCCCCAGGAGCAGGCCACAGACCCC CTTGTGGACAGCCTGGGCAGTGGCATTGTCTACTCAGCCCTTACCTGCCACCTG TGCGGCCACCTGAAACAGTGTCATGGCCAGGAGGATGGTGGCCAGACCCCTGTC ATGGCCAGTCCTTGCTGTGGCTGCTGCTGTGGAGACAGGTCCTCGCCCCCTACA ACCCCCCTGAGGGCCCCAGACCCCTCTCCAGGTGGGGTTCCACTGGAGGCCAGT CTGTGTCCGGCCTCCCTGGCACCCTCGGGCATCTCAGAGAAGAGTAAATCCTCA TCATCCTTCCATCCTGCCCCTGGCAATGCTCAGAGCTCAAGCCAGACCCCCAAA ATCGTGAACTTTGTCTCCGTGGGACCCACATACATGAGGGTCTCTTAG ANTI-HUMAN IL4Ra ANTIBODY SEQUENCES 69 VH EVQLVESGGG LEQPGGSLRLSCAGSGFTFR DYAMTWVRQA PGKGLEWVSS ISGSGGNTYY ADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKDR LSITIRPRYYGLDVWGQGTTVTVSS 70 VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQ LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTP YTFGQGTKLEIK 71 HEAVY CHAIN EVQLVESGGG LEQPGGSLRL SCAGSGFTFR DYAMTWVRQA PGKGLEWVSS ISGSGGNTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDR LSITIRPRYY GLDVWGQGTT VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL G 72 LIGHT CHAIN DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQ LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTP YTFGQGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSF HUMAN IGHG4 SEQUENCES 73 IGHG4*01 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG AMINO ACID VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV SEQUENCE ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV L HQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS R LTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK L  = position 189 R  = postion 289 74 IGHG4*01 GCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGA NUCLEOTIDE GCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTT SEQUENCE CCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACAC CTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCC TGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCT CATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGC CAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGG TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTA CCGTGTGGTCAGCGTCCTCACCGTC C TGCACCAGGACTGGCTGAACGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTA CACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCA G GCTCACCGTGGACAAG AGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGG CTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAA ATGA C  = position 565 (CTG encodes 189V) G  = position 866 (AGG encodes 289R) Nav1.7 SEQUENCES 75 HUMAN Nav1.7 MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPKEEKKDDDEEAPKPSSDLEAG AMINO ACID KQLPFIYGDIPPGMVSEPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYMLSPFS SEQUENCE PLRRISIKILVHSLFSMLIMCTILTNCIFMTMNNPPDWTKNVEYTFTGIYTFESLVKI (ENST00000303354) LARGFCVGEFTFLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFRVLRALKTISVI PGLKTIVGALIQSVKKLSDVMILTVFCLSVFALIGLQLFMGNLKHKCFRNSLENNET LESIMNTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPEGYTCVKIGRNPDYGYT SFDTFSWAFLALFRLMTQDYWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLIL AVVAMAYEEQNQANIEEAKQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIRRS RIMGLSESSSETSKLSSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKSESEDSIRR KSFHLGVEGHRRAHEKRLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGRGRDIGSETE FADDEHSIFGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLPVNGKMHSAVD CNGVVSLVDGRSALMLPNGQLLPEVIIDKATSDDSGTTNQIHKKRRCSSYLLSED MLNDPNLRQRAMSRASILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPYWIK FKKCIYFIVMDPFVDLAITICIVLNTLFMAMEHHPMTEEFKNVLAIGNLVFTGIFAA EMVLKLIAMDPYEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRSFRLLRVFKLA KSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKECVCKINDDC TLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLIVYMMVMVI GNLVVLNLFLALLLSSFSSDNLTAIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKA FSKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGHNFLKEKDKISGFGSSVDKHL MEDSDGQSFIHNPSLTVTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRSSSSE CSTVDNPLPGEGEEAEAEPMNSDEPEACFTDGCVWRFSCCQVNIESGKGKIWW NIRKTCYKIVEHSWFESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEM LLKWIAYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL RALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYECINTTD GSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIGVIIDNFNQQKKK LGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQAFDISI MVLICLNMVTMMVEKEGQSQHMTEVLYWINVVFIILFTGECVLKLISLRHYYFTV GWNIFDFVVVIISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVKGAKGIRTLLFA LMMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKKEDGINDMFNFETFGNSMICLF QITTSAGWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNPSVGIFYFVSYIIISFLV VVNMYIAVILENFSVATEESTEPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFA AALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLRSQ MEERFMSANPSKVSYEPITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISSIYIKD GDRDDDLLNKKDMAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKEKYEQDR TEKEDKGKDSKESKK 76 HUMAN Nav1.7 ATGGCAATGTTGCCTCCCCCAGGACCTCAGAGCTTTGTCCATTTCACAAAACA NUCLEOTIDE GTCTCTTGCCCTCATTGAACAACGCATTGCTGAAAGAAAATCAAAGGAACCCA SEQUENCE AAGAAGAAAAGAAAGATGATGATGAAGAAGCCCCAAAGCCAAGCAGTGACT (ENST00000303354) TGGAAGCTGGCAAACAGCTGCCCTTCATCTATGGGGACATTCCTCCCGGCATG GTGTCAGAGCCCCTGGAGGACTTGGACCCCTACTATGCAGACAAAAAGACTTT CATAGTATTGAACAAAGGGAAAACAATCTTCCGTTTCAATGCCACACCTGCTTT ATATATGCTTTCTCCTTTCAGTCCTCTAAGAAGAATATCTATTAAGATTTTAGTA CACTCCTTATTCAGCATGCTCATCATGTGCACTATTCTGACAAACTGCATATTT ATGACCATGAATAACCCACCGGACTGGACCAAAAATGTCGAGTACACTTTTAC TGGAATATATACTTTTGAATCACTTGTAAAAATCCTTGCAAGAGGCTTCTGTGT AGGAGAATTCACTTTTCTTCGTGACCCGTGGAACTGGCTGGATTTTGTCGTCA TTGTTTTTGCGTATTTAACAGAATTTGTAAACCTAGGCAATGTTTCAGCTCTTC GAACTTTCAGAGTATTGAGAGCTTTGAAAACTATTTCTGTAATCCCAGGCCTG AAGACAATTGTAGGGGCTTTGATCCAGTCAGTGAAGAAGCTTTCTGATGTCAT GATCCTGACTGTGTTCTGTCTGAGTGTGTTTGCACTAATTGGACTACAGCTGTT CATGGGAAACCTGAAGCATAAATGTTTTCGAAATTCACTTGAAAATAATGAAA CATTAGAAAGCATAATGAATACCCTAGAGAGTGAAGAAGACTTTAGAAAATA TTTTTATTACTTGGAAGGATCCAAAGATGCTCTCCTTTGTGGTTTCAGCACAGA TTCAGGTCAGTGTCCAGAGGGGTACACCTGTGTGAAAATTGGCAGAAACCCT GATTATGGCTACACGAGCTTTGACACTTTCAGCTGGGCCTTCTTAGCCTTGTTT AGGCTAATGACCCAAGATTACTGGGAAAACCTTTACCAACAGACGCTGCGTG CTGCTGGCAAAACCTACATGATCTTCTTTGTCGTAGTGATTTTCCTGGGCTCCT TTTATCTAATAAACTTGATCCTGGCTGTGGTTGCCATGGCATATGAAGAACAG AACCAGGCAAACATTGAAGAAGCTAAACAGAAAGAATTAGAATTTCAACAGA TGTTAGACCGTCTTAAAAAAGAGCAAGAAGAAGCTGAGGCAATTGCAGCGGC AGCGGCTGAATATACAAGTATTAGGAGAAGCAGAATTATGGGCCTCTCAGAG AGTTCTTCTGAAACATCCAAACTGAGCTCTAAAAGTGCTAAAGAAAGAAGAAC AGAAGAAAGAAAAAGAATCAAAAGAAGCTCTCCAGTGGAGAGGAAAAGGGA GATGCTGAGAAATTGTCGAAATCAGAATCAGAGGACAGCATCAGAAGAAAA AGTTTCCACCTTGGTGTCGAAGGGCATAGGCGAGCACATGAAAAGAGGTTGT CTACCCCCAATCAGTCACCACTCAGCATTCGTGGCTCCTTGTTTTCTGCAAGGC GAAGCAGCAGAACAAGTCTTTTTAGTTTCAAAGGCAGAGGAAGAGATATAGG ATCTGAGACTGAATTTGCCGATGATGAGCACAGCATTTTGGAGACAATGAG AGCAGAAGGGGCTCACTGTTTGTGCCCCACAGACCCCAGGAGCGACGCAGCA GTAACATCAGCCAAGCCAGTAGGTCCCCACCAATGCTGCCGGTGAACGGGAA AATGCACAGTGCTGTGGACTGCAACGGTGTGGTCTCCCTGGTTGATGGACGC TCAGCCCTCATGCTCCCCAATGGACAGCTTCTGCCAGAGGTGATAATAGATAA GGCAACTTCTGATGACAGCGGCACGACCAATCAAATACACAAGAAAAGGCGT TGTAGTTCCTATCTCCTTTCAGAGGATATGCTGAATGATCCCAACCTCAGACAG AGAGCAATGAGTAGAGCAAGCATATTAACAAACACTGTGGAAGAACTTGAAG AGTCCAGACAAAAATGTCCACCTTGGTGGTACAGATTTGCACACAAATTCTTG ATCTGGAATTGCTCTCCATATTGGATAAAATTCAAAAAGTGTATCTATTTTATT GTAATGGATCCTTTTGTAGATCTTGCAATTACCATTTGCATAGTTTTAAACACA TTATTTATGGCTATGGAACACCACCCAATGACTGAGGAATTCAAAAATGTACT GCTATAGGAAATTTGGTCTTTACTGGAATCTTTGCAGCTGAAATGGTATTAAA ACTGATTGCCATGGATCCATATGAGTATTTCCAAGTAGGCTGGAATATTTTTG ACAGCCTTATTGTGACTTTAAGTTTAGTGGAGCTCTTTCTAGCAGATGTGGAA GGATTGTCAGTTCTGCGATCATTCAGACTGCTCCGAGTCTTCAAGTTGGCAAA ATCCTGGCCAACATTGAACATGCTGATTAAGATCATTGGTAACTCAGTAGGGG CTCTAGGTAACCTCACCTTAGTGTTGGCCATCATCGTCTTCATTTTTGCTGTGG TCGGCATGCAGCTCTTTGGTAAGAGCTACAAAGAATGTGTCTGCAAGATCAAT GATGACTGTACGCTCCCACGGTGGCACATGAACGACTTCTTCCACTCCTTCCTG ATTGTGTTCCGCGTGCTGTGTGGAGAGTGGATAGAGACCATGTGGGACTGTA TGGAGGTCGCTGGTCAAGCTATGTGCCTTATTGTTTACATGATGGTCATGGTC ATTGGAAACCTGGTGGTCCTAAACCTATTTCTGGCCTTATTATTGAGCTCATTT AGTTCAGACAATCTTACAGCAATTGAAGAAGACCCTGATGCAAACAACCTCCA GATTGCAGTGACTAGAATTAAAAAGGGAATAAATTATGTGAAACAAACCTTA CGTGAATTTATTCTAAAAGCATTTTCCAAAAAGCCAAAGATTTCCAGGGAGAT AAGACAAGCAGAAGATCTGAATACTAAGAAGGAAAACTATATTTCTAACCAT ACACTTGCTGAAATGAGCAAAGGTCACAATTTCCTCAAGGAAAAAGATAAAA TCAGTGGTTTTGGAAGCAGCGTGGACAAACACTTGATGGAAGACAGTGATGG TCAATCATTTATTCACAATCCCAGCCTCACAGTGACAGTGCCAATTGCACCTGG GGAATCCGATTTGGAAAATATGAATGCTGAGGAACTTAGCAGTGATTCGGAT AGTGAATACAGCAAAGTGAGATTAAACCGGTCAAGCTCCTCAGAGTGCAGCA CAGTTGATAACCCTTTGCCTGGAGAAGGAGAAGAAGCAGAGGCTGAACCTAT GAATTCCGATGAGCCAGAGGCCTGTTTCACAGATGGTTGTGTATGGAGGTTCT CATGCTGCCAAGTTAACATAGAGTCAGGGAAAGGAAAAATCTGGTGGAACAT CAGGAAAACCTGCTACAAGATTGTTGAACACAGTTGGTTTGAAAGCTTCATTG TCCTCATGATCCTGCTCAGCAGTGGTGCCCTGGCTTTTGAAGATATTTATATTG AAAGGAAAAAGACCATTAAGATTATCCTGGAGTATGCAGACAAGATCTTCACT TACATCTTCATTCTGGAAATGCTTCTAAAATGGATAGCATATGGTTATAAAACA TATTTCACCAATGCCTGGTGTTGGCTGGATTTCCTAATTGTTGATGTTTCTTTG GTTACTTTAGTGGCAAACACTCTTGGCTACTCAGATCTTGGCCCCATTAAATCC CTTCGGACACTGAGAGCTTTAAGACCTCTAAGAGCCTTATCTAGATTTGAAGG AATGAGGGTCGTTGTGAATGCACTCATAGGAGCAATTCCTTCCATCATGAATG TGCTACTTGTGTGTCTTATATTCTGGCTGATATTCAGCATCATGGGAGTAAATT TGTTTGCTGGCAAGTTCTATGAGTGTATTAACACCACAGATGGGTCACGGTTT CCTGCAAGTCAAGTTCCAAATCGTTCCGAATGTTTTGCCCTTATGAATGTTAGT CAAAATGTGCGATGGAAAAACCTGAAAGTGAACTTTGATAATGTCGGACTTG GTTACCTATCTCTGCTTCAAGTTGCAACTTTTAAGGGATGGACGATTATTATGT ATGCAGCAGTGGATTCTGTTAATGTAGACAAGCAGCCCAAATATGAATATAGC CTCTACATGTATATTTATTTTGTCGTCTTTATCATCTTTGGGTCATTCTTCACTTT GAACTTGTTCATTGGTGTCATCATAGATAATTTCAACCAACAGAAAAAGAAGC TTGGAGGTCAAGACATCTTTATGACAGAAGAACAGAAGAAATACTATAATGC AATGAAAAAGCTGGGGTCCAAGAAGCCACAAAAGCCAATTCCTCGACCAGGG AACAAAATCCAAGGATGTATATTTGACCTAGTGACAAATCAAGCCTTTGATAT TAGTATCATGGTTCTTATCTGTCTCAACATGGTAACCATGATGGTAGAAAAGG AGGGTCAAAGTCAACATATGACTGAAGTTTTATATTGGATAAATGTGGTTTTT ATAATCCTTTTCACTGGAGAATGTGTGCTAAAACTGATCTCCCTCAGACACTAC TACTTCACTGTAGGATGGAATATTTTTGATTTTGTGGTTGTGATTATCTCCATT GTAGGTATGTTTCTAGCTGATTTGATTGAAACGTATTTTGTGTCCCCTACCCTG TTCCGAGTGATCCGTCTTGCCAGGATTGGCCGAATCCTACGTCTAGTCAAAGG AGCAAAGGGGATCCGCACGCTGCTCTTTGCTTTGATGATGTCCCTTCCTGCGT TGTTTAACATCGGCCTCCTGCTCTTCCTGGTCATGTTCATCTACGCCATCTTTGG AATGTCCAACTTTGCCTATGTTAAAAAGGAAGATGGAATTAATGACATGTTCA ATTTTGAGACCTTTGGCAACAGTATGATTTGCCTGTTCCAAATTACAACCTCTG CTGGCTGGGATGGATTGCTAGCACCTATTCTTAACAGTAAGCCACCCGACTGT GACCCAAAAAAAGTTCATCCTGGAAGTTCAGTTGAAGGAGACTGTGGTAACC CATCTGTTGGAATATTCTACTTTGTTAGTTATATCATCATATCCTTCCTGGTTGT GGTGAACATGTACATTGCAGTCATACTGGAGAATTTTAGTGTTGCCACTGAAG AAAGTACTGAACCTCTGAGTGAGGATGACTTTGAGATGTTCTATGAGGTTTGG GAGAAGTTTGATCCCGATGCGACCCAGTTTATAGAGTTCTCTAAACTCTCTGA TTTTGCAGCTGCCCTGGATCCTCCTCTTCTCATAGCAAAACCCAACAAAGTCCA GCTCATTGCCATGGATCTGCCCATGGTTAGTGGTGACCGGATCCATTGTCTTG ACATCTTATTTGCTTTTACAAAGCGTGTTTTGGGTGAGAGTGGGGAGATGGAT TCTCTTCGTTCACAGATGGAAGAAAGGTTCATGTCTGCAAATCCTTCCAAAGT GTCCTATGAACCCATCACAACCACACTAAAACGGAAACAAGAGGATGTGTCT GCTACTGTCATTCAGCGTGCTTATAGACGTTACCGCTTAAGGCAAAATGTCAA AAATATATCAAGTATATACATAAAAGATGGAGACAGAGATGATGATTTACTCA ATAAAAAAGATATGGCTTTTGATAATGTTAATGAGAACTCAAGTCCAGAAAAA ACAGATGCCACTTCATCCACCACCTCTCCACCTTCATATGATAGTGTAACAAAG CCAGACAAAGAGAAATATGAACAAGACAGAACAGAAAAGGAAGACAAAGGG AAAGACAGCAAGGAAAGCAAAAAATAG

TABLE 11 Human IL4Ra variants distributed over several human ethnic populations & having desired total human genotype frequency (a) Amino acid variability, population distributions and frequencies: Hom Freq⁴ No. No. Unique (Het + Hom Human Populations Individs¹ Pops² Het Freq³ freq⁵) Cum Freq⁶ Most 75I 835 0.462 0.302 0.533 common (0.764) Variant 75V ASW, CEU, CHB, CHS, 762 14 0.462 0.235 0.467 CLM, FIN, GBR, IBS, (0.697) JPT, LWK, MXL, PUR, TSI, YRI Most 400E 988 0.256 0.648 0.777 common (0.904) Variant 400A ASW, CEU, CHB, CHS, 384 14 0.256 0.095 0.223 CLM, FIN, GBR, IBS, (0.351) JPT, LWK, MXL, PUR, TSI, YRI Most 431C 1077 0.176 0.810 0.898 common (0.986) Variant 431R ASW, CEU, CHB, CHS, 207 14 0.176 0.014 0.102 CLM, FIN, GBR, IBS, (0.19) JPT, LWK, MXL, PUR, TSI, YRI Most 503S 1010 0.269 0.656 0.79 common (0.925) Variant 503P ASW, CEU, CHB, CHS, 376 14 0.269 0.075 0.21 CLM, FIN, GBR, IBS, (0.344) JPT, LWK, MXL, PUR, TSI, YRI Most 576Q 885 0.311 0.499 0.655 common (0.81) Variant 576R ASW, CEU, CHB, CHS, 547 14 0.311 0.190 0.345 CLM, FIN, GBR, IBS, (0.501) JPT, LWK, MXL, PUR, TSI, YRI Most 752S 1059 0.176 0.794 0.882 common (0.97) Variant 752A ASW, CEU, CHB, CHS, 225 14 0.176 0.030 0.118 CLM, FIN, GBR, IBS, (0.206) JPT, LWK, MXL, PUR, TSI, YRI Table Footnotes: ¹Number of individuals in 1000 Genomes database (20110521) found to have the allele; ²Number of unique human ethnic populations in 1000 Genomes database in which the allele was found to occur; ³Heterozygous human genotype frequency, ie, cumulative frequency of all genotypes having one occurrence of the variant allele and one occurrence of another allele (heterozygous state), eg, ac genotype in 1000 Genomes database; ⁴Homozygous human genotype frequency, ie, cumulative frequency of two occurrences of the variant allele (homozygous state), eg, cc genotype in 1000 Genomes database; and ⁵Total human genotype frequency, ie, total of heterozygous plus homozygous human genotype frequencies. ⁶Cumulative human allele frequency of all occurrences of the variant allele in 1000 Genomes database. (b) Nucleotide Sequence Variations of Selected Alleles Common Allele A A T T A T Variant Allele Nucleotide Position¹ 16: 27344882 16: 27362551 16: 27362643 16: 27362859 16: 27363079 16: 27363606 Non-Synonymous Nucleotide Variation² G C C C G G Variant ID³ rs1805010 rs1805011 rs1805012 rs1805015 rs1801275 rs1805016 Corresponding Amino Acid Variation 75V 400A 431R 503P 576R 752A Table Footnotes: ¹Notation is chromosome number (all positions are on human chromosome 1): coordinate number (Ensembl release 73 - September 2013, Genome assembly: GRCh37 (GCA_000001405.13), (forward strand); ²Nucleotide change (compared to most common allele) giving rise to an amino acid change in the variant form (compared to most common allele); and ³NCBI dbSNP reference number (NCBI dbSNP Build 138 released on Apr. 25, 2013).

TABLE 12 Human Nav1.7 (SNC9A) variants distributed over several human ethnic populations & having desired total human genotype frequency (a) Amino acid variability, population distributions and frequencies: Hom Freq⁴ No. No. Unique (Het + Hom Human Populations Individs¹ Pops² Het Freq³ freq⁵) Cum Freq⁶ VARIANTS ASSOCIATED WITH PRIMARY ERYTHERMALGIA (PE)⁷ Most 136V common Variant 136I Most 216F common Variant 216S Most 241S common Variant 241T Most 395N common Variant 395K Most 848I common Variant 848T Most 858L common Variant 858H 858F Most 863A common Variant 863P Most 1449F common Variant 1449V VARIANTS ASSOCIATED WITH PAROXYSMAL EXTREME PAIN DISORDER (PEPD)⁷ Most 996R common Variant 996C Most 1298V common Variant 1298F 1298D Most 1299V common Variant 1299F Most 1461I common Variant 1461T Most 1462F common Variant 1462V Most 1464T common Variant 1464I Most 1627M common Variant 1627K VARIANTS ASSOCIATED WITH CHANNELOPATHY-ASSOCIATED INSENSITIVITY TO PAIN (CIP)⁷ Most 277R common Variant 277X⁸ Most 328Y common Variant 328X Most 395N common Variant 395K Most 459S common Variant 459X Most 693E common Variant 693X Most 767I common Variant 767X Most 830R common Variant 830X Most 897W common Variant 897X Most 1200F common Variant 1200L Most 1235I common Variant 1235L Most 1488R common Variant 1488X Most 1659K common Variant 1659X Most 1689W common Variant 1689X FURTHER VARIANTS Most 422E 962 0.442 0.439 0.660 common (0.881) Variant 422D ASW, CEU, CHB, CHS, 613 14 0.442 0.119 0.340 CLM, FIN, GBR, IBS, (0.561) JPT, LWK, MXL, PUR, TSI, YRI Most 490S 1086 0.076 0.918 0.957 common (0.994) Variant 490N ASW, CEU, CHB, CHS, 89 14 0.076 0.005 0.043 CLM, FIN, GBR, IBS, (0.081) JPT, LWK, MXL, PUR, TSI, YRI Most 943M common Variant 943L ASW, CEU, CHB, CHS, 14 CLM, FIN, GBR, IBS, JPT, LWK, MXL, PUR, TSI, YRI Most 1161R 1076 0.189 0.797 0.891 common (0.986) Variant 1161W ASW, CEU, CHB, CHS, 222 14 0.189 0.015 0.109 CLM, FIN, GBR, IBS, (0.204) JPT, LWK, MXL, PUR, TSI, YRI Most 1919D 1076 0.109 0.876 0.931 common (0.985) Variant 1919G ASW, CEU, CHB, CHS, 135 14 0.109 0.015 0.069 CLM, FIN, GBR, IBS, (0.124) JPT, LWK, MXL, PUR, TSI, YRI Table Footnotes: ¹Number of individuals in 1000 Genomes database (20110521) found to have the allele; ²Number of unique human ethnic populations in 1000 Genomes database in which the allele was found to occur; ³Heterozygous human genotype frequency, ie, cumulative frequency of all genotypes having one occurrence of the variant allele and one occurrence of another allele (heterozygous state), eg, ac genotype in 1000 Genomes database; ⁴Homozygous human genotype frequency, ie, cumulative frequency of two occurrences of the variant allele (homozygous state), eg, cc genotype in 1000 Genomes database; and ⁵Total human genotype frequency, ie, total of heterozygous plus homozygous human genotype frequencies. ⁶Cumulative human allele frequency of all occurrences of the variant allele in 1000 Genomes database. ⁷See J Clin Invest. 2007 December; 117(12): 3603-9, “Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders”, Drenth J P & Waxman S G (incorporated herein by reference); amino acid position numbering is as per this reference. ⁸X here and elsewhere indicate change to an unknown amino acid (ie, a change from the most common amino acid at that position) Amino acid positions are as per Ensembl, unless otherwise indicated. (b) Nucleotide Sequence Variations of Selected Alleles Common Allele G T C C T T Variant Allele Nucleotide Position¹ 2: 166242648 2: 166198883 2: 166286469 2: 166272746 2: 166288485 2: 166277030 Non-Synonymous Nucleotide Variation² A C T A C G Variant ID³ rs6746030 rs3750904 rs58022607 rs4369876 rs13402180 rs12478318 Corresponding Amino Acid Variation 1161W 1919G 490N 1002L 422D 943L Table Footnotes: ¹Notation is chromosome number (all positions are on human chromosome 1): coordinate number (Ensembl release 73 - September 2013, Genome assembly: GRCh37 (GCA_000001405.13), (forward strand); ²Nucleotide change (compared to most common allele) giving rise to an amino acid change in the variant form (compared to most common allele); and ³NCBI dbSNP reference number (NCBI dbSNP Build 138 released on Apr. 25, 2013).

Example 4: Rarer IL6R Variants

The present invention provides anti-IL6R ligands; and IL6R-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of IL6R, in particular human IL6R or its ligands and in screening assays to identify other antagonists of IL6R activity. Some of the ligands of the invention are useful for inhibiting binding of IL6R to IL6 and/or gp130, or inhibiting IL6R-mediated activities.

Anti-IL6R ligands (eg, antibodies and anti-sense RNA) have been developed based on targeting and neutralising so-called “wild-type” human IL6R, which is a commonly-occurring form (see, eg, SEQ ID NO: 78). While such therapies are useful for human patients harbouring this form of human IL6R, the inventor considered it useful to investigate the possibility of targeting rarer—but still naturally-occurring—forms of IL6R amongst human populations. In this way, the inventor arrived at insight into the natural occurrences and distributions of rarer human IL6R forms that can serve as useful targets (at the protein or nucleic acid level) for human treatment, prophylaxis and diagnosis pertinent to diseases and conditions mediated or associated with IL6R activity. This particularly provides for tailored therapies, prophylaxis and diagnosis in humans that are devoid of the common IL6R gene or protein.

The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in activity and/or conformation of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to more effectively tailor medicines and diagnosis of patients. The invention, therefore, provides for tailored pharmaceuticals and testing that specifically addresses rarer IL6R polymorphic variant forms. Such forms or “alleles” (at the nucleotide level), comprise one or more changes at the nucleotide and amino acid levels from the corresponding common form nucleotide and amino acids sequences, ie, there are one or more non-synonymous changes at the nucleotide level that translate into one or more corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting such rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention.

With this realisation, the inventor realised that there is significant industrial and medical application for the invention in terms of guiding the choice of anti-IL6R ligand for administration to human patients for therapy and/or prophylaxis of IL6R-mediated or associated diseases or conditions. In this way, the patient receives drugs and ligands that are tailored to their needs—as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example the present inventor decided to determine a set of human IL6R variants on the basis of the following criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population. The inventor selected variants having at least 3 of the 4 following criteria:—

-   -   Naturally-occurring human IL6R variation having a cumulative         human allele frequency of 35% or less;     -   Naturally-occurring human IL6R variation having a total human         genotype frequency of about 50% or less;     -   Naturally-occurring human IL6R variation found in many different         human ethnic populations (using the standard categorisation of         the 1000 Genomes Project; see Table 14 below); and     -   Naturally-occurring human IL6R variation found in many         individuals distributed across such many different ethnic         populations.

On the basis of these criteria, the inventor identified the variants listed in Table 13 below. The inventor's selection included, as a consideration, selection for nucleotide variation that produced amino acid variation in corresponding IL6R forms (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.

TABLE 13 Human IL6R variants distributed over several human ethnic populations & having desired total human genotype frequency (a) Amino acid variability, population distributions and frequencies: Exon 9 Hom Freq⁴ No. No. Unique (Het + Hom Human Populations Individs¹ Pops² Het Freq³ freq⁵) Cum Freq⁶ Most 358D 0.383 0.489 0.680 common (0.872) Variant 358A YRI, ASW, GBR, TSI, 557 14 0.383 0.128 0.320 CLM, CHB, LWK, CHS, (0.511) MXL, PUR, JPT, IBS, FIN, CEU (See note 7) Most 385V 0.086 0.904 0.947 common (0.99) Variant 385I LWK, ASW, YRI, PUR 105 4 0.086 0.010 0.053 (see note 8) (0.096) Table Footnotes: ¹Number of individuals in 1000 Genomes database (20110521) found to have the allele; ²Number of unique human ethnic populations in 1000 Genomes database in which the allele was found to occur; ³Heterozygous human genotype frequency, ie, cumulative frequency of all genotypes having one occurrence of the variant allele and one occurrence of another allele (heterozygous state), eg, ac genotype in 1000 Genomes database; ⁴Homozygous human genotype frequency, ie, cumulative frequency of two occurrences of the variant allele (homozygous state), eg, cc genotype in 1000 Genomes database; and ⁵Total human genotype frequency, ie, total of heterozygous plus homozygous human genotype frequencies. ⁶Cumulative human allele frequency of all occurrences of the variant allele in 1000 Genomes database. 7. According to the 1000 Genomes database version 20130502, this variant is found in the following 26 human populations (KHV, GBR, CHB, CDX, CLM, MXL, CHS, JPT, GIH, PUR, ESN, FIN, ACB, BEB, YRI, ASW, ITU, PJL, TSI, PEL, MSL, LWK, STU, GWD, IBS, CEU) and in 1218 individuals with a cumulative frequency of 0.29. 8. According to the 1000 Genomes database version 20130502, this variant is found in the following 10 human populations (ASW, YRI, PEL, MSL, LWK, GWD, PUR, IBS, ESN, ACB) and in 267 individuals with a cumulative frequency of 0.06. (b) Nucleotide Sequence Variations of Selected Alleles Common Allele A G Variant Nucleotide Position¹ Allele 1: 154426970 1: 154427050 Non-Synonymous Nucleotide Variation² C A Variant ID³ rs2228145 rs28730736 Corresponding Amino Acid Variation 358A 385I Table Footnotes: ¹Notation is chromosome number (all positions are on human chromosome 1): coordinate number (Ensembl release 73 - September 2013, Genome assembly: GRCh37 (GCA_000001405.13); ²Nucleotide change (compared to most common allele) giving rise to an amino acid change in the variant form (compared to most common allele); and ³NCBI dbSNP reference number (NCBI dbSNP Build 138 released on Apr. 25, 2013).

Tailoring Antibodies to Rarer IL6R Variant Profile

As outlined above, the invention includes the possibility to tailor treatment of humans further by selecting antibody-based ligands with variable and/or constant domains based on gene segments found in many humans of the ethnic populations where the variant IL6R forms are found to meet the selection criteria of the invention. An example is provided for ligands comprising antibody VH domains derived from recombination of human IGHV gene segments comprising selected nucleotides at positions in the HCDR1 or FW3 where there is variability in humans (ie, where SNPs occur in humans).

The inventor analysed human IGHV variation and used this to choose ligands based on human IGHV alleles comprising said selected nucleotides and for matching to human recipient genotypes and/or phenotypes. The inventor identified utility in using gene VH gene segments encoding (i) a CDR1 comprising a Phe at position 4 shown in SEQ ID NO: 109 and wherein said human comprises a VH gene segment encoding a CDR1 comprising a Phe at position 4 shown in SEQ ID NO: 109, or the human expresses VH domains that comprise a CDR1 comprising a Phe at position 4 shown in SEQ ID NO: 109; or (ii) a FW3 comprising a Thr at position 33 shown in SEQ ID NO: 111 and wherein said human comprises a VH gene segment encoding a FW3 comprising a Thr at position 33 shown in SEQ ID NO: 111 or the human expresses VH domains that comprise a FW3 comprising a Thr at position 33 shown in SEQ ID NO: 111. Further information is provided in Table 14, which shows variation at these positions, as well as the variant distributions across the 1000 Genomes Project database relating to many human populations.

In other embodiments, as explained more fully above, the invention provides for ligands which are tailored to the human recipient's genotype and/or phenotype based on alternative human VH gene segments, or on Vκ, Vλ or constant region gene segments (see further Table 16 for representative variants).

In an example, following this guidance, the chosen ligand can be sarilumab.

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment, and wherein said human comprises a IGHV3-7*01 VH gene segment or the human expresses VH domains derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment. (ii) wherein the ligand comprises a Vκ domain derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment, and wherein said human comprises a IGKV1-12*01 Vκ gene segment or the human expresses Vκ domains derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment. (iii) wherein the ligand comprises a Vκ domain derived from the recombination of a human Vκ segment and a human Jκ segment, the human Vκ segment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 113 and wherein said human comprises a Vκ gene segment encoding a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 113, or the human expresses Vκ domains that comprise a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 113; or (ii) a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 115 and wherein said human comprises a Vκ gene segment encoding a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 115 or the human expresses Vκ domains that comprise a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 115. (iv) wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an Asp at position 204 shown in SEQ ID NO: 81 or a Leu at position 206 shown in SEQ ID NO: 81 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising an Asp at position 204 shown in SEQ ID NO: 81 or a Leu at position 206 shown in SEQ ID NO: 81. (v) wherein the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 83, an Asn at position 75 shown in SEQ ID NO: 83, a Phe at position 76 shown in SEQ ID NO: 83, a Val at position 161 shown in SEQ ID NO: 83 and an Ala at position 257 shown in SEQ ID NO: 83 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 83, Asn at position 75 shown in SEQ ID NO: 83, Phe at position 76 shown in SEQ ID NO: 83, Val at position 161 shown in SEQ ID NO: 83 or Ala at position 257 shown in SEQ ID NO: 83. (vi) wherein the ligand comprises a human kappa chain constant region that comprises a Val at position 84 shown in SEQ ID NO: 93 or a Cys at position 87 shown in SEQ ID NO: 93 and wherein said human comprises (i) an IGKC1*01 human kappa chain constant region gene segment, or the human expresses antibodies comprising human kappa chain constant regions comprising a Val corresponding to position 84 shown in SEQ ID NO: 93 or a Cys at position 87 shown in SEQ ID NO: 93. (vii) wherein the ligand comprises a human IGLC1*01 lambda chain constant region and wherein said human comprises (i) a human IGLC1*01 lambda chain constant region gene segment, or the human expresses antibodies comprising human IGLC1*01 lambda chain constant regions.

For example, as per example (iv), the inventor identified the possibility of addressing the rarer IGH-gamma-1 SNPs 204D (observed cumulative frequency of 0.296) and 206L (observed cumulative frequency of 0.283) individually or in combination. These residues are part of the CH3 domain, and as such they form part of antibody Fc regions. Thus, matching of these CH3 variations with the patient is especially beneficial for reasons as discussed above. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 81 or a Leu corresponding to position 206 of SEQ ID NO: 81 and wherein the genome of the human comprises a gamma-1 heavy chain constant region nucleotide sequence that encodes such an Asp or Leu or the human expresses antibodies comprising human gamma-1 constant regions comprising such an Asp or Leu. An example of such a ligand is sarilumab.

In another example, as per example (v), the inventor identified the possibility of addressing IGH-gamma-2 SNPs. This included consideration of Fc region variation—in this respect, the inventor focused on positions 161 and 257 which are in the Fc region. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro corresponding to position 72 of SEQ ID NO: 83, an Asn corresponding to position 75 of SEQ ID NO: 83, a Phe corresponding to position 76 of SEQ ID NO: 83, a Val corresponding to position 161 of SEQ ID NO: 83 and an Ala corresponding to position 257 of SEQ ID NO: 83; and wherein the genome of the human comprises a gamma-2 heavy chain constant region nucleotide sequence that encodes such a selected amino acid or the human expresses antibodies comprising human gamma-2 constant regions comprising such a selected amino acid.

In another example, as per example (vi), the inventor addressed human kappa constant region variation. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 93 or a Cys corresponding to position 87 of SEQ ID NO: 93; and wherein the genome of the human comprises a kappa light chain constant region nucleotide sequence that encodes such a Val or Cys or the human expresses antibodies comprising human kappa light chain constant regions comprising such a Val or Cys. An example of such a ligand is sarilumab.

In another example, as per example (vii), the inventor addressed human lambda constant region variation. Thus, in this example the ligand of the invention comprises or consists of an antibody that comprises a human IGLC2*01 light chain constant region; and wherein the genome of the human comprises a human IGLC2*01 nucleotide sequence or the human expresses antibodies comprising human light chain IGLC2*01 constant regions.

Determination of Specific Binding of Ligands of the Invention to IL6R Variants

The specific binding of ligands of the invention to IL6R variants can be performed using the following method.

Method of SPR Determination of Binding Binding of the antibodies to the IL6R variants is carried out by SPR using the ProteOn XPR36™ Array system (BioRad). An anti-human IgG surface (Jackson Labs 109-005-008) was created on a GLC Biosensor chip by primary amine coupling. Test antibodies are captured on this surface as ligands. The IL6R variants are used as analytes and passed over the captured antibodies at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. Binding curves are double referenced using a buffer injection (i.e. 0 nM) to remove baseline drift and injection artefacts. Regeneration of the capture surface is with 100 mM phosphoric acid which removes the captured antibody allowing another cycle of capture and binding. The binding sensorgrams generated are analysed using the 1:1 model inherent to the ProteOn XPR36 Array system analysis software. The assay is performed at 25° C. and using 1×HBS-EP (Teknova) as running buffer.

REFERENCES

The references cited herein are incorporated by reference in their entirety

-   1. Ferreira et al (PLoS Genet. 2013 April; 9(4):e1003444. doi:     10.1371/journal.pgen.1003444. Epub 2013 Apr. 4, “Functional IL6R     358Ala allele impairs classical IL-6 receptor signaling [sic] and     influences risk of diverse inflammatory diseases”; Rantala A et al,     Hum Immunol. 2011 January; 72(1):63-8. doi:     10.1016/j.humimm.2010.10.010. Epub 2010 Oct. 15, “Association of     IL-6 and IL-6R gene polymorphisms with susceptibility to respiratory     tract infections in young Finnish men”; -   2. Zhang H Y et al, Oral Dis. 2014 January; 20(1):69-75. doi:     10.1111/odi.12075. Epub 2013 Feb. 24, “The association of IL-6 and     IL-6R gene polymorphisms with chronic periodontitis in a Chinese     population”; -   3. J C Galicia et al, Genes and Immunity (2004) 5, 513-516.     doi:10.1038/sj.gene.6364120 Published online 12 Aug. 2004,     “Polymorphisms in the IL-6 receptor (IL-6R) gene: strong evidence     that serum levels of soluble IL-6R are genetically influenced”; -   4. Esparza-Gordillo J et al, J Allergy Clin Immunol. 2013 August;     132(2):371-7. doi: 10.1016/j.jaci.2013.01.057. Epub 2013 Apr. 9, “A     functional IL-6 receptor (IL6R) variant is a risk factor for     persistent atopic dermatitis”.

TABLE 14 1000 GENOMES PROJECT HUMAN POPULATIONS Below is a summary of the ethnic populations as per the 1000 Genomes Project sequences. (a) 100 Genome Populations Population Code Population Description Super Population Code CHB Han Chinese in Bejing, China ASN JPT Japanese in Tokyo, Japan ASN CHS Southern Han Chinese ASN CDX Chinese Dai in Xishuangbanna, China ASN KHV Kinh in Ho Chi Minh City, Vietnam ASN CEU Utah Residents (CEPH) with Northern and EUR TSI Western European ancestry EUR Toscani in Italia FIN Finnish in Finland EUR GBR British in England and Scotland EUR IBS Iberian population in Spain EUR YRI Yoruba in Ibadan, Nigera AFR LWK Luhya in Webuye, Kenya AFR GWD Gambian in Western Divisons in The Gambia AFR MSL Mende in Sierra Leone AFR ESN Esan in Nigera AFR ASW Americans of African Ancestry in SW USA AFR ACB African Carribbeans in Barbados AFR MXL Mexican Ancestry from Los Angeles USA AMR PUR Puerto Ricans from Puerto Rico AMR CLM Colombians from Medellin, Colombia AMR PEL Peruvians from Lima, Peru AMR GIH Gujarati Indian from Houston, Texas SAN PJL Punjabi from Lahore, Pakistan SAN BEB Bengali from Bangladesh SAN STU Sri Lankan Tamil from the UK SAN ITU Indian Telugu from the UK SAN (b) Super Populations AFR, African AMR, Ad Mixed American ASN, East Asian EUR, European SAN, South Asian (c) Population Ancestries European ancestry Utah residents (CEPH) with Northern and Western European ancestry (CEU) Toscani in Italia (TSI) British from England and Scotland (GBR) Finnish from Finland(FIN) Iberian populations in Spain (IBS) East Asian ancestry Han Chinese in Beijing, China (CHB) Japanese in Toyko, Japan (JPT) Han Chinese South (CHS) Chinese Dai in Xishuangbanna (CDX) Kinh in Ho Chi Minh City, Vietnam (KHV) Chinese in Denver, Colorado (CHD) (pilot 3 only) West African ancestry Yoruba in Ibadan, Nigeria (YRI) Luhya in Webuye, Kenya (LWK) Gambian in Western Division, The Gambia (GWD) Malawian in Blantyre, Malawi (MAB) West African Population (TBD) Americas African Ancestry in Southwest US (ASW) African American in Jackson, MS (AJM) African Caribbean in Barbados (ACB) Mexican Ancestry in Los Angeles, CA (MXL) Puerto Rican in Puerto Rico (PUR) Colombian in Medellin, Colombia (CLM) Peruvian in Lima, Peru (PEL) South Asian ancestry Ahom in the State of Assam, India Kayadtha in Calcutta India Reddy in Hyderabad, India Maratha in Bombay India Punjabi in Lahore, Pakistan

TABLE 15 Exemplary anti-IL6R disclosures, eg of antibodies and/or antibody fragments, assays, treatments, formulations, kits, methods and indications, useful in any and all aspects of the invention Patent or patent application which is incorporated by reference in its entirety, and specifically, eg, with respect to the SEQ ID Nos. comprising an anti-IL6R monoclonal antibody or fragment thereof U.S. Pat. No. 8,568,721, US20130157313A1, US20130149310A1, US20130122003A1, U.S. Pat. No. 8,192,741, U.S. Pat. No. 8,183,014, US20120003697A1, U.S. Pat. No. 8,080,248, U.S. Pat. No. 8,043,617, US20110171241A1, US20100316636A1, US20100316627A1, U.S. Pat. No. 7,582,298

TABLE 16 Human antibody gene segment variants distributed over several human ethnic populations-useful for ligand tailoring Example Nucleotide Human Amino Acid Variation³ Hom Gene Example Coordinate² (NCBI dbSNP Variant Freq⁸ Segment Human & reference Nucleotide Human No. Het (Het + Hom Cum Type Allele¹ Variation number)⁴ Position Populations⁵ Individs⁶ Freq⁷ freq⁹) Freq¹⁰ IGHV3-9 IGHV3-9*01 30F TTT (IMGT numbering) (Phe at position 4 in SEQ ID NO: 32) (CDR1 variation) IGHV3-9*02 30S TCT (IMGT numbering) (CDR1 variation) IGHV3-9*01 110T ACG 14:106552310 B 1015 0.082 0.847 0.888 (Thr at position 33 in (forward strand) (0.929) SEQ ID NO: 34) (FW3 variation) IGHV3-9*03 110M ATG 14:106552310 B  167 0.082 0.071 0.112 (FW3 variation) (rs8020204) (forward strand) (0.153) IGKV3-11 IGKV3-11*01 115P CCT 2:89326669 B 1090 0.064 0.934 0.966 (Pro at position 7 in (forward strand) (0.998) SEQ ID NO: 36) (CDR3 variation) 115H CAT 2:89326669 B   72 0.064 0.002 0.034 (CDR3 variation) (rs182958807) (forward strand) (0.066) IGKV3-11*01 87S TCT 2:89326754 B 1090 0.074 0.924 0.961 (Ser at position 15 in (forward strand) (0.998) SEQ ID NO: 38) (FW3 variation) 87P CCT 2:89326754 B   83 0.074 0.002 0.039 (FW3 variation) (rs191612627) (forward strand) (0.076) IGKC IGKC*01 84V GTC 2:89156948 B  454 0.345 0.071 0.243 (Val at position 84 in (forward strand) (0.416) SEQ ID NO: 16) IGKC*04 84L CTC 2:89156948 B 1015 0.345 0.584 0.757 (rs232230) (forward strand) (0.929) IGKC*01 87C TGC 2:89156939 (Cys at position 87 in (forward strand) SEQ ID NO: 16) IGKC*02 87G GGC 2:89156939 (r5s200765148) (forward strand) IGHG1 IGHG1*01 204D GAT 14:106208086 A  153 0.400 0.096 0.296 (CH3 variation) (forward strand) (European (0.496) ancestry) IGHG1*03 204E GAG 14:106208086 A  366 0.400 0.504 0.704 (CH3 variation) (rs1045853) (forward strand) (European (0.904) ancestry IGHG1*01 206L CTG 14:106208082 A    0.358 0.104 0.283 (CH3 variation) (forward strand) (European (0.462) ancestry) IGHG1*03 206M ATG 14:106208082 A    0.358 0.538 0.717 (CH3 variation) (rs11621259) (forward strand) (European (0.896) ancestry) IGHG2 IGHG2*01 72P CCC 14:106110914 B    0.336 0.540 0.708 (CH1 variation) (forward strand) (0.876) IGHG2*02 72T ACC 14:106110914 B    0.336 0.124 0.292 (CH1 variation) (rs11627594) (forward strand) IGHG2*01 75N AAC 14:106110904 A    0.007 0.993 0.997 (CH1 variation) (forward strand) IGHG2*04 75S AGC 14:106110904 A    0.007 0.004 (CH1 variation) (rs201590297) (forward strand) IGHG2*01 76F TTC (CH1 variation) IGHG2*04 76L TTG (CH1 variation) IGHG2*01 161V GTG 14:10611013 B    0.342 0.539 0.711 (CH2 variation) (forward strand) (0.881) IGHG2*02 161M ATG 14:10611013 B    0.342 0.118 0.289 (CH2 variation) (rs8009156) (forward strand) (0.46) IGHG2*01 257A GCC 14:106109752 C    0.199 0.493 0.592 (CH3 variation) (forward strand) (0.692) D 0.007 0.992 0.995 (0.999) IGHG2*06 257S TCC 14:106109752 C    0.199 0.308 0.408 (CH3 variation) (rs4983499) (forward strand) (0.507) D 0.007 0.002 0.005 (0.009) Table Footnotes: 10. IMGT notation (ww.imgt.org); refer to figures for other alleles comprising this variation. 11. Numbering as indicated in Ensembl (available on the World Wide Web at ensembl.org) unless otherwise indicated 12. SNP Underlined in Codon. 13. NCBI dbSNP Build 138 released on Apr. 25, 2013. 14. Human population used for representative variant frequency analysis. Populations A This population included 662 participants of European descent from the ClinSeq project, all of whom had undergone whole-exome sequencing using Agilent's 38 Mb or 50 Mb capture kit. B 1000 Genomes database. C ESP6500:African American. D ESP6500:European American. 15. Number of individuals in representative population found to have the allele. 16. Heterozygous human genotype frequency, ie, cumulative frequency of all genotypes having one occurrence of the variant allele and one occurrence of another allele (heterozygous state), eg, ac genotype in the population. 17. Homozygous human genotype frequency, ie, cumulative frequency of two occurrences of the variant allele (homozygous state), eg, cc genotype in the population. 18. Total human genotype frequency, ie, total of heterozygous plus homozygous human genotype frequencies. 19. Cumulative human allele frequency of all occurrences of the variant allele in the population.

TABLE 17 SEQUENCES SEQ ID Human NO: Allele Nucleotide/Amino Acid Sequence  78 IL-6R MLAVGCALLAALLAAPGAALAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPED NATVHWVLRKPAAGSHPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHL LVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQ EPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQP DPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMV KDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAE NEVSTPMQALTTNKDDDNILFRDSANATSLPVQ D SSSVPLPTFLVAGGSLAFGT LLCIAI V LRFKKTWKLRALKEGKTSMHPPYSLGQLVPERPRPTPVLVPLISPPV SPSSLGSDNTSSHNRPDARDPRSPYDISNTDYFFPR D  = position 358 V  = position 385  79 ATGCTGGCCGTCGGCTGCGCGCTGCTGGCTGCCCTGCTGGCCGCGCCGGGAGCG GCGCTGGCCCCAAGGC GCTGCCCTGCGCAGGAGGTGGCGAGAGGCGTGCTGACCAGTCTGCCAGGAGACA GCGTGACTCTGACCTG CCCGGGGGTAGAGCCGGAAGACAATGCCACTGTTCACTGGGTGCTCAGGAAGCC GGCTGCAGGCTCCCAC CCCAGCAGATGGGCTGGCATGGGAAGGAGGCTGCTGCTGAGGTCGGTGCAGCTC CACGACTCTGGAAACT ATTCATGCTACCGGGCCGGCCGCCCAGCTGGGACTGTGCACTTGCTGGTGGATG TTCCCCCCGAGGAGCC CCAGCTCTCCTGCTTCCGGAAGAGCCCCCTCAGCAATGTTGTTTGTGAGTGGGG TCCTCGGAGCACCCCA TCCCTGACGACAAAGGCTGTGCTCTTGGTGAGGAAGTTTCAGAACAGTCCGGCC GAAGACTTCCAGGAGC CGTGCCAGTATTCCCAGGAGTCCCAGAAGTTCTCCTGCCAGTTAGCAGTCCCGG AGGGAGACAGCTCTTT CTACATAGTGTCCATGTGCGTCGCCAGTAGTGTCGGGAGCAAGTTCAGCAAAAC TCAAACCTTTCAGGGT TGTGGAATCTTGCAGCCTGATCCGCCTGCCAACATCACAGTCACTGCCGTGGCC AGAAACCCCCGCTGGC TCAGTGTCACCTGGCAAGACCCCCACTCCTGGAACTCATCTTTCTACAGACTAC GGTTTGAGCTCAGATA TCGGGCTGAACGGTCAAAGACATTCACAACATGGATGGTCAAGGACCTCCAGCA TCACTGTGTCATCCAC GACGCCTGGAGCGGCCTGAGGCACGTGGTGCAGCTTCGTGCCCAGGAGGAGTTC GGGCAAGGCGAGTGGA GCGAGTGGAGCCCGGAGGCCATGGGCACGCCTTGGACAGAATCCAGGAGTCCTC CAGCTGAGAACGAGGT GTCCACCCCCATGCAGGCACTTACTACTAATAAAGACGATGATAATATTCTCTT CAGAGATTCTGCAAAT GCGACAAGCCTCCCAGTGCAAGATTCTTCTTCAGTACCACTGCCCACATTCCTG GTTGCTGGAGGGAGCC TGGCCTTCGGAACGCTCCTCTGCATTGCCATTGTTCTGAGGTTCAAGAAGACGT GGAAGCTGCGGGCTCT GAAGGAAGGCAAGACAAGCATGCATCCGCCGTACTCTTTGGGGCAGCTGGTCCC GGAGAGGCCTCGACCC ACCCCAGTGCTTGTTCCTCTCATCTCCCCACCGGTGTCCCCCAGCAGCCTGGGG TCTGACAATACCTCGA GCCACAACCGACCAGATGCCAGGGACCCACGGAGCCCTTATGACATCAGCAATA CAGACTACTTCTTCCC CAGATAG HEAVY CHAIN ALLELES  80 IGHG1*01 gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctggg (CH1 + Hinge + ggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcg CH2 + CH3 + CH-S) tggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctca ggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacc tacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagccc aaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggggga ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccct gaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactgg tacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaac agcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaag gagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctcc aaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgag ctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatc gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtg ctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtgg cagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacg cagaagagcctctccctgtctccgggtaaa  81 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR D E L TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK D  = position 204 L  = position 206  82 IGHG2*01 gcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgag (CH1 + Hinge + agcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcg CH2 + CH3 + CH-S) tggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctca ggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacc tacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgc aaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttc ctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgc gtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggc gtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgt gtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgc aaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaaggg cagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaac caggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgg gagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgac ggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctc tccctgtctccgggtaaa  83 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTV P SS NF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG V EVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDI A VEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK P  = position 72 N  = position 75 F  = position 76 V  = position 161 A  = position 257  84 IGHV3-9*01 gaagtgcagctggtggagtctgggggaggcttggtacagcctggcaggtccctg agactctcctgtgcagcctctggattcacct t tgatgattatgccatgcactgg gtccggcaagctccagggaagggcctggagtgggtctcaggtattagttggaat agtggtagcataggctatgcggactctgtgaagggccgattcaccatctccaga gacaacgccaagaactccctgtatctgcaaatgaacagtctgagagctgaggac a c ggccttgtattactgtgcaaaagata t  = nucleotide number 86 c  = nucleotide number 272  85 EVQLVESGGGLVQPGRSLRLSCAASGFT F DDYAMHWVRQAPGKGLEWVSGISWN SGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAED T ALYYCAKD F  = position 30 (IMGT nomenclature) T  = position 110 (dbSNP nomenclature)  86 IGHV3-7*01 gaggtgcagctggtggagtctgggggaggcttggtccagcctggggggtccctg agactc tcctgtgcagcctctggattcacctttagtagctattggatgagctgggtccgc caggct ccagggaaggggctggagtgggtggccaacataaagcaagatggaagtgagaaa tactat gtggactctgtgaagggccgattcaccatctccagagacaacgccaagaactca ctgtat ctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaga ga  87 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQD GSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR  88 IGHJ3*02 T GAT GCT TTT GAT ATC TGG GGC CAA GGG ACA ATG GTC ACC GTC TCT TCA G  89 D A F D I W G Q G T M V T V S S  90 IGHJ6*01 AT TAC TAC TAC TAC TAC GGT ATG GAC GTC TGG GGG CAA GGG ACC ACG GTC ACC GTC TCC TCA G  91 Y Y Y Y Y G M D V W G Q G T T V T V S S LIGHT CHAIN ALLELES  92 IGKC*01 cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatct ggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacag tggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggac agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgag aaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaag agcttcaacaggggagagtgt  93 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHK V YA C EVTHQGLSSPVTKSFNRGEC V  = position 84 C  = position 87  94 IGLC1*01 cccaaggccaaccccacggtcactctgttcccgccctcctctgaggagctccaagccaac aaggccacactagtgtgtctgatcagtgacttctacccgggagctgtgacagtggcttgg aaggcagatggcagccccgtcaaggcgggagtggagacgaccaaaccctccaaacagagc aacaacaagtacgcggccagcagctacctgagcctgacgcccgagcagtggaagtcccac agaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccct acagaatgttca  95 PKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETT KPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS  96 IGKV1-12*01 gacatccagatgacccagtctccatcttccgtgtctgcatctgtaggagacagagtcacc atcacttgtcgggcgagtcagggtattagcagctggttagcctggtatcagcagaaacca gggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatca aggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcct gaagattttgcaacttactattgtcaacaggctaacagtttccctcc  97 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP  98 IGKV3-11*01 gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccacc ctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacct ggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagcc aggttcagtggcagtggg t ctgggacagacttcactctcaccatcagcagcctagagcct gaagattttgcagtttattactgtcagcagcgtagcaactggc c tcc t  = nucleotide number 199 c  = nucleotide number 284  99 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSG S GTDFTLTISSLEPEDFAVYYCQQRSNW P S  = position 87 P  = position 115 100 IGKJ2*01 tgtacacttttggccaggggaccaagctggagatcaaac 101 YTFGQGTKLEIK 102 IGKJ4*01 G CTC ACT TTC GGC GGA GGG ACC AAG GTG GAG ATC AAA C 103 L T F G G G T K V E I K 104 An IGHG1*01 EVQLVESGGG LVQPGRSLRL SCAASRFTFD DYAMHWVRQA PGKGLEWVSG Heavy Chain ISWNSGRIGY ADSVKGRFTI SRDNAENSLF LQMNGLRAED TALYYCAKGR DSFDIWGQGT MVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVTYL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 105 An IGKC*01 DIQMTQSPSS VSASVGDRVT ITCRASQGIS SWLAWYQQKP GKAPKLLIYG Kappa Light ASSLESGVPS Chain RFSGSGSGTD FTLTISSLQP EDFASYYCQQ ANSFPYTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 106 An IGHG1*01 EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMSWVRQAPGKGLEWVAKISPG Heavy Chain GSWTYY SDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLWGYYALDIWGQGTT VTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 107 An IGKC*01 EIVLTQSPATLSLSPGERATLSCSASISVSYMYWYQQKPGQAPRLLIYDMSNLA Kappa Light SGIPAR Chain FSGSGSGTDFTLTISSLEPEDFAVYYCMQWSGYPYTFGGGTKVEIKRTVAAPSV FIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 108 IGHV3-9*01 ggattcacct t tgatgattatgcc CDR1 (IMGT t  = position 11 nomenclature) 109 G F T  F  D D Y A F  = position 4 110 IGHV3-9*01 ggc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc FW3 (IMGT aga gac aac gcc aag aac tcc ctg tat ctg caa atg aac nomenclature) agt ctg aga gct gag gac a c g gcc ttg tat tac tgt c  = position 98 111 G Y A D S V K G R F T I S R D N A K N S L Y L Q M N S L R A E D  T  A L Y Y C T  = position 33 112 IGKV3-11*01 CAG CAG CGT AGC AAC TGG C C T CC CDR3 (IMGT C  = nucleotide 20 Nomencalture) 113 QQRSNWP P = position 7 114 IGKV3-11*01 aacagggccactggcatcccagccaggttcagtggcagtggg t ctgggacagacttcact FW3 (IMGT ctcaccatcagcagcctagagcctgaagattttgcagtttattactgt Nomencalture) T  = nucleotide 43 115 NRATGIPARFSGSG S GTDFTLTISSLEPEDFAVYYC S  = position 15 127 Aflibercept SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDS polypeptide RKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTII

sequence

FVRVHEKDKTHTCPPCP A PELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKA K

QPREPQVYTLPPSR D E L TKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG (SEQ ID NO: 127) DKTHTCPPCP (SEQ ID NO: 128) = C-terminal part of IGHG1*01 hinge A  = start of human IGHG1*01 CH2 sequence K  = end of human IGHG1*01 CH2 sequence

 = start of human IGHG1*01 CH3 sequence D  = Asp204 (corresponding to position 204 in SEQ ID NO: 42) L  = Leu 206 (corresponding to position 206 in SEQ ID NO: 42) Flt1 Ig-like C2-type 2 domain GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEAT VNGH (SEQ ID NO: 129) KDR Ig-like C2-type 3 domain Y

 (SEQ ID NO: 130) 129 Flt1 Ig-like GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEAT C2-type 2 VNGH domain 130 KDR Ig-like YDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGS 02-type 3 EMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNST domain 131 A human VEGFA MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVD sequence IFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEM (isoform 206) SFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPG PHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR 132 A human VEGFA MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVD sequence IFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEM (isoform 165) SFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELN ERTCRCDKPRR 133 A human PD-L1 MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALI sequence VYWEME (ENST00000381577) DKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRC MISYGG ADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQV LSGKTT TTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHP PNERTH LVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET 134 A human IL-2 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL sequence TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIV LELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 135 Nav1.8 AMINO MEFPIGSLETNNFRRFTPESLVEIEKQIAAKQGTKKAREKHREQKDQEEKPRPQLDLKAC ACID SEQUENCE NQLPKFYGELPAELIGEPLEDLDPFYSTHRTFMVLNKGRTISRFSATRALWLFSPFNLIR RTAIKVSVHSWFSLFITVTILVNCVCMTRTDLPEKIEYVFTVIYTFEALIKILARGFCLN EFTYLRDPWNWLDFSVITLAYVGTAIDLRGISGLRTFRVLRALKTVSVIPGLKVIVGALI HSVKKLADVTILTIFCLSVFALVGLQLFKGNLKNKCVKNDMAVNETTNYSSHRKPDIYIN KRGTSDPLLCGNGSDSGHCPDGYICLKTSDNPDFNYTSFDSFAWAFLSLFRLMTQDSWER LYQQTLRTSGKIYMIFFVLVIFLGSFYLVNLILAVVTMAYEEQNQATTDEIEAKEKKFQE ALEMLRKEQEVLAALGIDTTSLHSHNGSPLTSKNASERRHRIKPRVSEGSTEDNKSPRSD PYNQRRMSFLGLASGKRRASHGSVFHFRSPGRDISLPEGVTDDGVFPGDHESHRGSLLLG GGAGQQGPLPRSPLPQPSNPDSRHGEDEHQPPPTSELAPGAVDVSAFDAGQKKTFLSAEY LDEPFRAQRAMSVVSIITSVLEELEESEQKCPPCLTSLSQKYLIWD0CPMWVKLKTILFG LVTDPFAELTITLCIVVNTIFMAMEHHGMSPTFEAMLQIGNIVFTIFFTAEMVFKIIAFD PYYYFQKKWNIFDCIIVTVSLLELGVAKKGSLSVLRSFRLLRVFKLAKSWPTLNTLIKII GNSVGALGNLTIILAIIVFVFALVGKQLLGENYRNNRKNISAPHEDWPRWHMHDFFHSFL IVFRILCGEWIENMWACMEVGQKSICLILFLTVMVLGNLVVLNLFIALLLNSFSADNLTA PEDDGEVNNLQVALARIQVFGHRTKQALCSFFSRSCPFPQPKAEPELVVKLPLSSSKAEN HIAANTARGSSGGLQAPRGPRDEHSDFIANPTVWVSVPIAEGESDLDDLEDDGGEDAQSF QQEVIPKGQQEQLQQVERCGDHLTPRSPGTGTSSEDLAPSLGETWKDESVPQVPAEGVDD TSSSEGSTVDCLDPEEILRKIPELADDLEEPDDCFTEGCIRHCPCCKLDTTKSPWDVGWQ VRKTCYRIVEHSWFESFIIFMILLSSGSLAFEDYYLDQKPTVKALLEYTDRVFTFIFVFE MLLKWVAYGFKKYFTNAWCWLDFLIVNISLISLTAKILEYSEVAPIKALRTLRALRPLRA LSRFEGMRVVVDALVGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFWRCINYTDGEFSL VPLSIVNNKSDCKIQNSTGSFFWVNVKVNFDNVAMGYLALLQVATFKGWMDIMYAAVDSR EVNMQPKWEDNVYMYLYFVIFIIFGGFFTLNLFVGVIIDNFNQQKKKLGGQDIFMTEEQK KYYNAMKKLGSKKPQKPIPRPLNKFQGFVFDIVTRQAFDITIMVLICLNMITMMVETDDQ SEEKTKILGKINQFFVAVFTGECVMKMFALRQYYFTNGWNVFDFIVVVLSIASLIFSAIL KSLQSYFSPTLFRVIRLARIGRILRLIRAAKGIRTLLFALMMSLPALFNIGLLLFLVMFI YSIFGMSSFPHVRWEAGIDDMFNFQTFANSMLCLFQITTSAGWDGLLSPILNTGPPYCDP NLPNSNGTRGDCGSPAVGIIFFTTYIIISFLIMVNMYIAVILENFNVATEESTEPLSEDD FDMFYETWEKFDPEATQFITFSALSDFADTLSGPLRIPKPNRNILIQMDLPLVPGDKIHC LDILFAFTKNVLGESGELDSLKANMEEKFMATNLSKSSYEPIATTLRWKQEDISATVIQK AYRSYVLHRSMALSNTPCVPRAEEEAASLPDEGFVAFTANENCVLPDKSETASATSFPPS YESVTRGLSDRVNMRTSSSIQNEDEATSMELIAPGP 136 Nav1.8 AAAAGTGACTGGAAACAGTGAGTAGAAGTAAGTCCTTTATTAAAAGCCAATAGAGAACAG NUCLEOTIDE GAGAGAGGAGGGAAGACCTGGATGGGCTCTGCATCAAGGGAAAGTTTTTAAAGTTGGAGG SEQUENCE AAAGTTAAGCATCTTTAAATGCTGATAGGAAAGAGCTGGCAGACAAGGAGAGAGAAAAAA ATGAGGTAGATATGAGATAAGATCCTAAGCAGAGGGAAAGGAATGGTCCTTAGAATGTAG GAAGATACCTCTCATGTTGTAAGAGAAGGGAAGGAGGAAAGAAGGCTGCAGACACAGGTG AGTTGGAGGTTTGATGGTATGAAGTTGGCAGAGTTCTGTTTTTCTCCATGAACTAGGAAA TATGGTCATTGGCTTACTGCAAGGGAAGACAGGGAATAGTGAGAAAAGAGTGTATTTGAA ACCTCAGACTAGAAGAAACTGCTGACTAGGGATACCATATGATTCCCAGGCAATACTGAG GTTCCCCATAACCCCTCAGCAGTTCAGGGGAACTGTTCTAGATTGACTTAGCCTTGGGAT TTAGGATAAGGGTGAGGGAGTGGCAAAATTGTGGACCACAGGTCTAATCTGGTTATGGGG AAAAGTGTTCAGCCACAGACTGAACCTGGAGGTCCCAGAGAAATTGGAGGTCCCAATGAA ATTCTCCAATAATCTGGAGTTCTCCAGTAACTTGGAGGTCCAAATGAAATTCAAACAGGT GGAGAAATGTTGACAATGTCAATAAATAAAAGATTGTGATAAGAAAGTAGCATGAGACTT AAGATAAAGTAGACTATAAACCAAAGTCTTTGATGAATATGGGAGAGTGCCTGGAACTCA GTGGTCCTGGAAATGAGGAGAGAGGGCAGCATGGCTAAATCTGGTGGTGGTGGTAGGATG GTACAAAGTGGTGAAGAAGCAATGACATAGAAGCACATGGGGAGCAAAGACATAACCCTG AGGCACCTGGAGGAGGGTTGAATAAACAGCCTCCCCTGAATGCCTTGCAGGGGAATGGGT TCCTGGGAGGAGCCAAGTGTGAGTTCAGGGAGGGGCCAGATATGAGGGTGGGAGAAGGGC TGTTCTGACAATCAAGATGGGACAGATGAGAGGGACAGGGCCAGTGGGCAAGCTGTCACC TCTCTGTGGTTATGTCCACTCTTATAAGAGTATAAATACTTCCTGAAGAAGAATGAGAAG ATGGAATTCCCCATTGGATCCCTCGAAACTAACAACTTCCGTCGCTTTACTCCGGAGTCA CTGGTGGAGATAGAGAAGCAAATTGCTGCCAAGCAGGGAACAAAGAAAGCCAGAGAGAAG CATAGGGAGCAGAAGGACCAAGAAGAGAAGCCTCGGCCCCAGCTGGACTTGAAAGCCTGC AACCAGCTGCCCAAGTTCTATGGTGAGCTCCCAGCAGAACTGATCGGGGAGCCCCTGGAG GATCTAGATCCGTTCTACAGCACACACCGGGTAAGAGCTACTAGTAGGAATGTCTGCTCT CAGCTCTTGGAGAGGAAGTCTCGGTCCCACCCAGCCCAGAAAGTCCTCCTTGGCCCTGGG TGATGTGGCAGTCTGCTAAAGATTCCGTTAACAACCAAAAAAAATATATATATATATCTC TAACTTATTTGATGATCTCTTTGAATTTTTATGACATTTATGATAGAAACTAACTAAACC CACATCTTCAAAAGCAGATAACTGCTGCAGAGCTTTCAGGGAAAGTTGGCTGTTCCCTTG GATGACCTCTGGCCTGGGGCCTCCAGAGGAGTTGGGTCTCTTTGCAGGTGGAGAGCAGAC ATCTTGAGGTTGGGGGAGGAGCATGGCGGTGAGGACAGAAAGCAGGATGAATTTAAGCCA GTGGTCCTCAACTATGATCTTGGAACCCCTGGTGTCCCCAAAACTGTTTTAGTGGGTTTA TAAAATGAAAACTATTGTATAACAATACTTGGATGTAATTTGTCTTTTTCACACACTTTC TCTTATGATCATACATTGGTGAATATAGAGGCTATTGATGGGGGATGATGTCATCACTCT AAAAGCTACTGGAATGTGTGCCTGTATATTCCCGTGTTTTAAAATTTCCTGAGTTTTGAA TTTCTAATATGAGATATATATACACACACATATGATGTATATATGTGTGTATACATCTAT ACACTATATATATACATACAACTATGTATATACATACAACTAGACCATGTAGCTAGTTAT GTATATATAGGTATATAAGCATATATACATATATATTAATTTCTAATATGATATACATAT ATCTGGCATAAACAAAAAGGTATCAACATAAACAAAAGTTATTTGGGGTCCTCAATAAGG TTTGAGAGTAATAATCCCTGAAATCAAAAAGTTTGAGAATCAGTAGTTTAAACAAGAGCT TCTTCATGACAATTATCTCACCTGTGTTGTATTTTTGATGTTCTACGAGCCAAAAAATGG TGAATGCATGTTTGGGAGTTAGGAGGACAGGAATCAGCCATCTCAAATATGGCTAGCAGA GTAAATAAAAAGGCACTGAATATCTTTCTTCCTCCTCTCCCATGCCAAGAGTTCATATCC TCCTAAATCCTCAGAGGGAGAAACCAGGAGAGGATTTAAGAGACAGAGATGTAGAAGGTG GAGACAGAAGAAACATTACAAAACAAGGATGGTTCTCTTGTCAGAAGAGAAATTAAATGC AACCATAACTTCACCATCCTGTTGCCAGAGTCCTCCCCTGGGCATAACACCTCTTACCAG CCTGAGGATTGGCTGTCTGTCCATTAAAGTACCCTCAGTGGGCTCATCTTGGTGCACTGT CAGTCCCAGAGATGAGGATCTTGCTGAGAGGGTGAGGATATGGCTTTTAGTCCTTGATCC TCTCTGCCTCTCTGTACCTGCATTTGACTCTCATTGACCTGTAGTTCCCAGAGCCCTTCT TGCTCATAAGCCTGAGATAATGCCTCTCATGTTTAAAAACATGGTTGTATAAGGAATCTT GTTTCTCATTGGAGGTCCACTTTCTGTTTTGTTTTGTTTCAGACATTTATGGTGCTGAAC AAAGGGAGGACCATTTCCCGGTTTAGTGCCACTCGGGCCCTGTGGCTATTCAGTCCTTTC AACCTGATCAGAAGAACGGCCATCAAAGTGTCTGTCCACTCATATCCTTTGCACGATTGC CTCTTCCACGTGTTACAATTAGACCTTGCCTACTGTGTCCAGTACCTTCTTTAGAGGTTA GCAACAGAGCCCTTATCCAATTGAATCCCAAAAGTACAATGAGGTCATTAGATTGAAAAA ATGGCTTGGGCCTTTAATGTGTCCCACACCCCAAAGCAGTCCCAAGACACAGAAGAAGTG TTTATGCAACACACAGGTGGTTTGGATTCACCTCAGACTAATCTAGACACACCTGTGGAT GCACAACTTGAAAAACATCCTCTTTATGAGGGGAGGTTTGCTGATGCCCCCAGACACATT CTCACCACGGTGTGTGGTCCGCTTTCCCCACAACACAACAGGCAGTAAGAGGGCCCAAGC CTTCTTTGGGCAGATGAGATAGGCATGAGTTGTTGCTCTGTGCCATAGCCATGCTGAGAA GTGGCTGCTTACTGGGCAAAAGAGCAGCACCTCCCAGAGGGCAAGGAAAACAGACCATGA TGGGAAGAGCACACATAGTCTCAATGGGACCTCATTGGTCATGGGGCACTGTTAACATCA CAAATTACTCAAAAACTACTAGCAGGTTTAAAATGTCAATAATCTGATGTTCATGCCATA CAAGACTGTAATGATTAGAGACACATTGGTCAAATGTCTACTATATGAATTGTTTTAGGT AATGTCAGAAGCTGCTTTCAGGGGATTTTGCAAGGCAAGCCAAACTAGTAGAAAAAAGAT ACTTTGGTTTGGTTTCCTAAGATCTTCCTGGGTGATCCTAATGGAAAGATCCTAATGACC TAAGATCCTTGGTCATCGGAAGGATTGGAAGTTCTTTATGACAAATTTAGCCCTGGCTTG CAGGAGGTTGATGTACAAGAATACACCTGCCTCAGTTGCCTGACAAATGGTAACCAGCCT TTTCTTAGAAGGCTTCAGTAAAAAAGAGTTCTACCCTGCTCCCTAACTCTGAGGCAGATT GTTGGATTTCTGGACAGTTGTCATTGAGCTGGTAACCTTGGCACAGTCATCTATCTGCAC ACTCAACATGAAGCTGCACAGCTTGATGAAGTTGCATTACGTTTTCTCTTTTTACATTTT TTTCTTTCTGGCTGGCCAGCAAGAATTACGTTTTCTGAGCTGAATCTTGAATTTTCAGAT TCTACAGAAGGATATTTAATGACAATACAGTCTCTCACTTGCTAAATACTTCCTAAGTGT CAATACCATCATCAGTTCAAAAGCCTCTGAAGTAGAGATTACCATCCCCATTTTACAGAT AATGAAACTGACACACAGAGGCATTAAGTAAATTATCCAAGACCATGTAGCTAGTTAAGA AGTCCAGCCTGCATCCTGCGTAGAGCAGTATCTACAGTGAGGAAGTAGGGTGGAAAGTAA TCAGGGTAATTATTCTCTCAATGTGTCGTCTCTGCTTGGGTAGTTGTTGCTTTTTCAAAA TATATACAATTACATGGAAATTACAAATAAAGAGAAATCACAAACATACAAACTCCTCTT TATGGATTCTGGTTGAATTACATCTGGATAAGTAAGAAACAGCTAATTAGAAATAAAATC CCCTAAAAAGAACCAAGTGACCAGTGAATATTCTCTTCATCTTGAGATTTGATGAGTAAA TAATTGCTTGGTAGAGTCACAGCTGGACAGAAAAGCTGTTGAGGGGGTCTCTTCCTCACA AAAGTCTGCATTTAAATCTCATAAATTTGGTAACTCCAGCCCCAAAAGATACTAGAATTC TCAGGGCCCTATTATTAATATTTCACCAAAAATATAATTCTAATCACATTTGCTATGAAT GTTTTCATAACTAATATTATACATAAGATATATTACTTTTAAATATTTGTAGAATTGGAG ACACGATTTTAGTTTAATCCTTGGATTAACATTTCCCCCCATGTGAGCATATTTCTTAAT TTAATTGTAAGTCAGCAGGAAAATGTGGTTTAATTAATAGAATTTTCCTTTTCATAATAG ATGTGTTCCTAAGTAACTGTAAAACTAGACACATCAAGGCATTTACAGAAAACTAAAATA CATTTAAATACTTCTGCTCAAATTGAAAAATGAGTGTTGTACTCTCTCTGTAGGTCAAGT TTATCTTCAAGTTTTATTTTTCCTTAGGAAGCACTGTTGGGAGCCCAGCTATTCCTAAGA AAAATGTCCAAGTGGGAACAATTTACCTTTAGATTCTCCCCACCAACATTTCTTCCCTTC TATTTTCCCCTTCCACCCACCTTCCTTTCAATGTCTCCCTCCCCTTTTGATAATCCCTTT GCCCATAGTCTGCTGTCAATATTCTCCACTCTAGCTATTCAGAGTTCTCCACTCCAGCCT AATCCTACTTGGGGTTTTCTTCCTGAGCCAGTATTTCATTTCCCTGTGCCTGCCTCCACC GTTCCTACCCAGAAGACCATTTCCTCGGTGGAACTGATCTCAACTAAGTTACCCAAACAA TCTGATGAAACTAATTCCCATGAAAGTTTGTACCTCTTCACTTATCTCCAAGTTTCTAAC TTTTCTTTGTCTTTTAGAATAAAAAAGACATTTGCCAGATGAAATTTCAGGCCATTTTGT ATACTGAAATGAAGGCTTCGGGGCAGAAGGACATTTTGGAAAAGGGAGTCTGTTTCCTTC TATCTTGCTCAGAGTTTGTTATCAAACAGATAAGAGTTCACATCTCTGCAGCATGGTTGA CTAGCTGCATGACCTGAGAAAAGTTATTTAACTTTTATTTTTTGTCAGCCTGTTTCTCAG CTTCAAAATGTAGATATTTATACCCACCTGTAGATCAGTTGTATTAGAAGTAACATGGAC AAAGTACTCAGCCTAGCTTAGGCACTCAGTAAATGTTAGTGGTTATTATGTGACAAGATC ATAAAGCTAATATTGGCTAAGATTTATGAATGTAATCATTCAGCATCAAGGTGATCCTAG ATGACATCACAGAGACTTGGTGATCTCAGCTTTTCCTTAACACAGGCCTTTCAGGTGGTT CAGTTTATTTATTACGGTCACTATTTTGGTTAATTGTGTGTGCATGACCCGAACTGACCT TCCAGAGAAAATTGAGTGAGTAGATTTGCTATCATTATTGTACCATCTCTTTTTGCTTTC TTTTCTTCCTTCACTTGGCAGTCTTTGTCTTTTATTTATCCTGCCATGCCTCATGTGGGT AATGCTGAATCTCATTGTCTATATGTCTTTGTAATAACCAGGTCTCTCCTTCTGTGAGAT TTGTTTCATAAACATACATGATTTTCAGTTTCAAAGAGATACAGATTAGTGGTTCTCAAC TTTGGCTGCATATTGCAATTTTTGGGGAGATTTTTTAAATGCCTGAGTCCCAGCCCCAGA AAGTCTAAGGTGGCCTGGGAAGATGAGTCTCCTCGCCCCCACCCCCCCCCAACACACACA CACTCACTCACCTGCTAGATGCACACACAAATGTATGCCTTAGAGAAGGTATGCTTTTGA GAAGGCCTGCTATGAAAGAGAGACAGGAGCAAAACTGAGACAGAACTAAGAACAAATGGC TCTGGAGTATATTTAGAAAATAACTCATAAATGAAGCTGTTTAAGTTATCATGCTTCATA TGCCTCAAAGTAACCTTAGTATTCCTGAAGTTATTCTATTTCCCTAGGTTAGTGGGTGAA ACCCCTATAGTTTCAGAATCTGGGAGTTTTCCAAAAACATTCCATATCCACACATCTGGG AGGGATGCTTCCACTCCAGCTTGAACCTAACATACCTTTGGTTATAATAACAGGTGCTTT GCAGAGCTTTCTTTTTTTTTTTTTTTTTTTTTTTGCTAATCATAACAGTAATGCATGCCA CAACAGAAAATACGTTATATTTTTATTAATAGATTAAAGGAAAAGGATCACATAGTCTTC TTAATTGATGCTAAAATCATTTGATAAAGTTTAATTTCCATTTATAATTTTAAATTCTTA GAAAAGTTGAAATAGAAGGGAATAAATGTGGCACATATACACCATGGAATGCTATGCAGT CATAAAAAAGGATGAGTTCATGTCCTTTGCAGGGACATGGATGAAGCTGGAAACCATCAT TCTCAGCAAACTAACACAAGAACAGAAAACCAAACACCGTATGTTCTCACTCATAAGTGG GAGTTGAACAATGAGAACACATGGACACAGGGAGGGGAACACACTGGGGCCTGTTGAGGG GTGGGGGGCTAGGGGAGGGATAGCATTAGGAGAAATACCTAATGTAAATGACGGGTTGAA GGGTGCAGCAAACCACCATGGCACGGGTACACCTATGTAACAAACCGGCACGTTCTGCAC ATGTACCCCAGAACTTAAAGTATAATAATTTTAAAAAGGGGATTGCATCACTCTGATAAA GGGTATTCATTGTTTTAAAGTATCAAGCGTCATACTTAATTGTGAACATGGAAAGCTTTC CCTTTGAGATCAAAAGAAAAAAAAACCAAAGATGTCTGTTCTTCTCAGTTCCACTTTGTA AAGGAGGTCTCAGCCAGCATAGTAAAGCAAAGAAAAAAAAAGTGCAAGAAGAAAAAAGTA CCGAAGATGTAATTTACAGATAATAAGATTGTATACATAAAATATCCAACATAACCTAGA GATAAACAGTAAGAATTAATAAAAGAGTATAGTAAGGTAGCTGGGTACAAATTAACATAC ATAAATCAATTGTGTATAGACACTAGCAACAAACAGAAAATAGTTTTTAAAAATACCATT TACACTGGCATTTTAAAATGTCAAGTATCTAGGAATAAGATTATCAAAGTATAAGTGAGA TCTCTATAAGGAAAATTATAATAAGTATTGTGTAAAGTTTTTAAAGAAAACCAAATCAGT GGGGAGACATGTTCTCAGATTCAAAGTCTCAATATTGTAAAGACTCCCAAAAATGATTAA TAAATTTAATATGATCCTCAAATCAATATTTCAACAGGCTTCTTTTGGTATTACTTGACA AACTTTAAACTTTAAATTTTCTTCCTCAAAAGTTTACATGAAATTTTAAAGAGCAAATTA TAGCCAAGAAACTTTTGAGGAAGAAATACAAAGTGGGAGAACTCATTCTACTGGATGTCG AGATTTACTATAAAGCTCTAATTGAAGCAGTGTGCCATTGGTGTAAGATTGGTCAGAATA ATGTAACAGCATAGACAGTCCATAAACAGACCAACACATATGTAAACATCTAATGTGTGA AAAGGTGGCACCCCAGAACAGCAGGGAAAAGGCGGTCTTCAAAAAATGATGCTGAAGAAT TAGGAATCTCACTGGAAAAACTACATTAAACATCCAACTTTGCACCACACACAAGAATCA ATTCTAGGTGGATCAACACCTCACTTGCTTTGTGAAATGCAAATATATAAAGCCCTTTTT TAAATTTTACTTTAAGTTCTGGGGTACATGTGCAGAAAGTGCAGGTTTGTTACATAGGTA TACACATGTCATGGTGGTTTGCTGCACCCATCAACCCATCATCTACATTAGGTATTTCTC CTAATGCTATCCCTCCCCTAGCCCCCAACCCCTCAACAGGCCCTGGTGTGTGATGTCCCC CTCCCTGTATCCACGTGCTCTCATTGTTCATCTCCCACCTATGAGTGAGAACATGCGGTG TTTGGTTTTCTGTTCTTATGTCAGTTTGCTGAGAATGATGGTTTCCAGCTTCATCCATGT CCCTGCAAAGAACATGAACTCACCCTTTTTTATGGCTGCATAGTATTCCATGGTGTATAT GTGCCATATTTTCTTTATCCAGTCTATCACTGATGGGCGTTTGGGTTAGTTCCAAGTCTT TGCTATTGTCAATAGTGCCACAATAAACATACGCATGCTTGTATCTTTATAGTAGAATAA TTTATAATCCTTTGGGTATATACCCAGTAATGGAATTGCTGGGTCAAATGGTGTTTCTGG TTCTAGATCCTTGAGGAATCACCACACTGTCTTCCACAATGGTTGAACTAATTTACACTC CCACCAACAGTGTAAAAGCATTCCTATCTCTCCACATCCTCTCCAGCATCTGTTGTTTCC TGACTTTTTAATGATCTCCATTCTAACTGGTATGAGACTCCATCTCATTGTGGTTTTGAC TGGCATTTCTCTAATGACCAGTGATGATGAGCTTTTTATCATATGTTTATTGGCTGCATA AATGTCTTCTTTTGAGAAGTGTCTGTTCATATCCTTTGCCCACTTTTTGATGGAGTTGTT TTTTCTTGTAAATTTGTTTAAGTTTTTTGTAGATTCTGGATATTAGCCCTTTGTCAGATT GATAGATTGCAAAAATTTTCTCCCATTCTGTAGGTTACCTATTCACTCTGATGATAGTTT CTTTTGCTGTGCAGAAGCTCTTTAGTTTAATTGGATTCCATTTGTCAATTTTGGCTTTTG TCACCATTGCTTTTGATGTGTTAGTCATAAAGCCTTTGCCCATGCCTATGTCCTGAAGGG TATTGCCTAGGTTTTCTTCTAGGGTTTTTATGGTTTTAGGTCTTATGTTTAAGTCTTTAA TCTATCTTGAGTTAATTTTTGTGTAAGGTATAAGGAAGGGATCTAGTTTCAATTTTCTGC CTAAGGCTAGCCAGTTTTCCCCACACCATTTATTAAATAGGGAATCCTTTCCCCATTGCT TGCTTTTGTCAAGTTTGTCAAAGATCAGATGGTTGTAGATATGCGGTGTTACTTCTGAGG CCTCTGTTCTGTTCCATTGGTCTAGATATCTGTTTTGGTACCAGTACCTCGCTGTTTTGG TTACTGTAGCTTTGTAGTATAGCTTGAAGTCAGGTAGCGTGATGCCTCCAACTTTCTTCT TTTTGCTTAGAATTGTCTTGACTATGCGGGCTCTTTTTTGTTCCACATGAAATTTAAAGT AGATTTTTCTAATTCTGTGAAGAAAGTCAGTGGTAGCTTGATGGGGATAACATTGAATCT ATAAATTACTTTGGGCAGTATGGCCATTTTCACAATATTGATTTTTCCTATCCATGAGCA TGGAATGTTTTTCCATTTATTTGTGTCCTCTCTTATTTCCTTGAGCAGTGATTTTTAGTT CTCCTTGAAGAGGTCCTTCACATCCCTTGTAAGTTGTATTTCTAGATATTTTATTCTCTT TGTTGCAATTGTGAATGGGAGTTCACTCATGATTTGGCTCTCGGTTTGTCTGTTATTGGT GTATGGGAATGCTTGTGATTTTTGCACATTGATTTTGTATCCTGAGACTTTGCTGAAGTT GCTTATCAGCTTAAGGAGATTTTGGGCTGAGATGATGGGGTTTTCTAAATATACAGTCAT GTCATCTGCAAACAGAGACAATTTGACTTCCTCTTTTCCTATTTGAATACCCCTTATTTC TTTCTCTTGCCTGATCGCCCTGGCCAGAACTTCCAATACTATGTTGAATAAGAGTGGTGA GATAGGGCATTCTTGTCTTGTGCTGGTTTTCAAAGGAAATGCTTCCAGTTTTTGCCCATT CAGTATGATATTGGCTGGGGGCTTGTCATAAATAGTTCTTATTATTTTGAGATACATTCC ATCAATACCTAGTTTATTGAGAGCTTTTAGCATGAATGGGTGTTGAATTTTGTCGAAGAC CTTTTCTGCATCTATTGAGATAATCACGTGGTTTTTGTCATTGGTTCTGTTTGTGTGATG GATTACGTTTATTGACTTGCGTATGTTGAACCAGCTTTGATCCCAGGTATGAAGCCGACT TGATCGTGGTGGATAAGCTTTTTGAAAGGAAAAAACAAAGAGGAATATCTTTATGACCTC AGACTAAGAAAAACTTTATTACATATGACACAAGAAGCACTAACTATACAGGAAAAAACT GATACATTTTAGTATACTATAATTAAGAATGTTCATCAGAAGACAACTTTAAGAGCATGA AAAAGCAAGTGCTTATATTCAGAGTAATGAACTCTACAAATCAATTAGAAAAATGTCAGA ACAACCACTTGTAAAATAGCACAAAATTTGAATAGGCACTTTACAAAAGTGGAAATCCAA ATGGCCATAAACATATGAAAGTATTCTTAACCTTTTTTTAAATCAAAGAGTTGCAAATTA AAATCACACTGAGATTTTATTACATGTTCATCATAGAAGGGAAATTTTTTAAAGTGTCAC AATACCAAGTATTGGGAATGACATGGAGCAACAGAAACTCACACACTGCTGGTGGGAGTG TAAATTTGGACAACCTCTTTGGAAAGCAAGGGGTATTATCTAGTAAAGGTGTAAGCATAC ATATGCTGTATTCTTCTATTACACCCTTAGGTACCTATGTTTAACAGAAATATGTACTTG TGGACACCAGATTACAGCTACAAGAAAGTTTATGAACATTTGCCCATAAAAACCCCAAAC CGGAAACCACTCAAATGTCCATAAACTTTGAGTACATTAACTATTTCTGTTATATTCTAA AATGAGAAAACTTACAGCAATAAAAATTAATAAACTTTAGTGCTGCACAACAATAAGGAT GAGTCTTAAGAAAGTAATATTGAACAAAAAAAGCCAAATGCAAAGGATTACATAAACAAG GTTCATTTATATAAAGTTTGAAATCAAGCAAAACTAAACTATTTTGTTTAGGAATGCATA CTTATGAAGTAAAAAGCATCAATAAAGTAAGGCAAGAAAAAGTACCAAAAATTAGGAGAA TAGTGGCCTCCAGGGCAGAAGAGGGAGGATGTAATTGGGAAGGAGCTGGTGAAGTGCTGG TCACGTTCCATTTTATGGCATGGGTAGTTGTCACACAAGCTGCTATGGAGTGAATTTTGT CCCCTCAAAATTCAAATGTTGAAACAAACCCCCCATGTTGCTATGTTGGAGATTGGGCCT CTAAGGAGGTGATTGGGTTAAATGAAGTCACAAGAGTAGAGCCCTTAAGTGGTAGAACTG GTGTCCTTATAAGAGAAAGAGACAGCTTTCTCCCCTTTGTGTGAGAACACAAGGACAAGG TAGACATCTATAAGCTAGAAAGAGAGTCTTTCCCAGACCCAACTATGCTGACATCCCAAT CTCAGGCTTCAACCTCCAGATTTGTGAGAAAATAAATTTCTGTTGCATAAGCCACCCAGT CTATGGTATTTTGTTAAGGCAACAAAGGCTGACTAATACACAAACATTTGTTTTAAAATA ATTAATTTATGTTTTATGTACATTTTTATTATGCATGTTACATATCATAATAAAAACCCA ATAATTTTTTAAAATAAATGACCATTGCAAAAAATGCAGAACACTGAAAATATAAATCAG AAGATGGTTACTAAGTAGAGAAATCACAAAATCATGAAATTATATTTAATATTGTTGAGA TCATATAGCATGTAAAATGTCTATGCTTCTTTTGGAGTTTTAATGTAAAACATTTTTTTG CAAAATGAGTTATTTGGGAGTTATTTGGAGATTGTGGTTAAATATGGAAGATAGAATGCA TATGTTTTACCCCACCCTCTCAAAATGCCAATAGAATGACAGCACGGGGGAAAGAACAGT ATAAATATACAAGAACAAAGAGAAAGAGAATGGAAAACACAGAGGACAAATGATGTCAAA ACACTTGGAAGACCAAAAGTGGGTGGAGAAGTGGTAAATCACTTAGCAGATCAGGGTTTG TTAAACCACGGTGCCTCCAGAGGGGGATAAAAATGAGAATAAAGGCATTTTACCGCCCCC GCTGCCCAACAAGCCCCAGAGGCCCTGCAAGTGTTGACATGATACAAATAGCAAGAAATG GAAGCAGAAAGAGGAAAGAACATGTGAGCAGAGTATGTTCACTTACTGCTGCTTCTATAA TAACAAGAAGTTAAAGAGTATTATTTAAAGTTGACAAATAAAGAAATGGACAAAATAATT ACACTTGACAGTACAAAGGTAAGCACAAAAAGATATTATTAAATACAGTCAGATAATGGA TAAAACAGAGAGGAAAGGAATCACCAAATGGAAATGAGAGATTTTGATTAAAGAAATAGT ACACTAAGGACAATGTATTACACGGTTTTAATTATAATTATAAATTTATAATAAGTTTTT ACTTATAAAGACAACCACTAGGATAAAGATACAAACTCTTTAGTAATCTATATATTTGTA ACAAATAATATGACAAACTCTAAATACTGGAAGAATTGTACATCAAAAAATAAAAAGCAA ATAAGAGATGCCAAATAATGAAAAGGTACAAAAGTAATTGGGGTTTCAGACCATAAATAT TAAATCATTATAACTAGGCTCAAACACATCTTTATTAATCAAAGTAGGAACCATTACAAT CAACACATTTTTGCCAATGAGAAATGAGTTTGTTTATTCCTGTAGCATAAAAATCCATGC TTTGGGATTCAATGAACTCTTGGAAGGCATTTTCTGCATCCCGCTGGTTATGGAAGTGTT TCACCTGCAAAAAGTTGTTCAGATGCTTGAAGAAGTGGTAGTTGGTTGGTGAGAGGTCAG GTGAATATGGTGGATGAGGCAAAACTTCATAGCCCAATTCATTCAACTTTTGAAGTAACT GTTGGTTGTGTGACATGCAGTCGGTTGTTGTGGAGAAGAATTGGGCCCTTTCTGTTGACC AATGCCAGCTGCAGGCCTTGCAGTTTTCTGTGCATCTCATCGATTTGCTGAACATATTTC TCAGATGTAATGGCTTTGCTGGGATTCAGAAAGCTGTAGTAGATCAGACCGGCAGCAGAC CACCAAGCAGTGACCACGACTTTTTTTGGTGCAAGTTGGGTTTTGGGAAGTGCTTTGGAG CTTCTTCTCAGTCCAGCCACCAAGCTGGTCATTGCCAGTTGTCAAGTAAAATTCACTTTT TATCGCACATCACAATCTGATTAAGAAATAGTTCATTTTTGTTGCATAGAATAAGAGACG ACAACACTTCAAAATGACGATTTTTTAAACATTTTTCCTTAGCTTATGAGGCACCCATTT ATGGAGCTGTTTCACCTTTCCAATTTGCTTCAACTGCCGAATGACCGTAGAATGGTCAAC GATGAGTTCTTCCTCAGCTTCTTGTGCAGTTGTAAGAGGATCAACTTCGATGATTGCTCT CAATTGGTCATTGTCAACTTCCAATGGCCAGCCACTACTCTCCTCATCTTCAAGGTTCTC ATCTCCTTTGCAAAACTTCTTGAACCACCACTGCACTGTACCTTTGTTAGCAATCCCTGG GCCAAATGCGTTGTTGATGTTGAGTTGTCTCTGCTGCTTTACAACCCATTTTGAACTCAA ATAAGAAAATCGCTCGAATTTGCTTTTTGTATAACATAATTTCCATAGTCTAAAATAAAT ATAAAATAAACAGCAAGTAATAAGTCATTAGCAAAAAAAAAAGTGAGAAATGCACATTAA AATGATGTATAACATGGTCACATTTATTTAAGAATGTATTCCAATATCAAACGGCAAATT TCAACAATGCAAAAACCACAATTACTTTTGTACCAACCTAATATTTTAAAAACTAAATAC GTCATATGAAATAATATAAACAAGCTAAGACTAAGCAAATCTGTCATGTCACTAAATATA AATGGATTTTACTCACCTATTTTTAAAAATGCCTATTACATTACAAAGCAATACCCAACT CTATGTTGAATAAAAAAAATACTCCTCACGGTGATACAGAAAGGCTAAAATTATAGAATT CAAAAGACATAAGCAAAGGCAAACAAAGAAAGCAGGATCCATGATTTTAATATCAGAGAA AGTGGAATCTCGGTCAAAAAGCATGAAATAGGACAAAAATTGCACAGACATATTAATATT AAAGACGATAGTAATCTATATCTTTGCAACAAATAAGATGGCAACAATATTTATAAATCA GAAATTATTAGAGTAGGAGGATAAATAAAAACATTGTAGGAATAGAAATTTTCAATTAAT TTTAGCCAATAACAGATCAAATGTATTAAAAAGTATGTAAAATTATAGATGACCTAGTAA TATAATTAATATAATGTATCTGATTAATATATAGCAAACTACCTTCCCTAACAATAGAAA ATGCACCTCCTTTTCAAAAAACCAAACATATCAAACCAAATATTAAGCCATGACAAAAAC CTCAATAAGCTCCAGAAAGTGGAAATAATATAGAAAACATTCTCTGATCATAATGAAATA GAGTTAAAAAAAATAACCAAATTTAATGAAATGAGAGGACTTTCCACTTGATAATTTTTA AAAATCTCTCTTAAAGAATTCTTGGACAAAAGACAAACTATAAACTAAAATTATATAATT TCGAGGGAAGGAAAGATAAAAAACAGTTTAATCAGAACCTAGAGAGTGCAGTTAAAGACA TACATATTCAGGGAATAAGTCACAGCATGGAAGCATAGAATATTTATATCAGTAAAAGCT GAAAAGAAATTATTTAAAGACCTAACAAAACTAGAAAAAGGATAACAAATGAAAGAAAGA AGTTAATAATTATAAAAAGCAATAAATTACAAAATAGAAAAACAGTAGATCTAATAAATC CAAATGCTGTTGTAAAAATATGCACAAGACCTTTATACTAAAAAATAATACAGCATTATT GAGAGAAATTTTAAAGTACCTAAATGAATTAAAGGATGTCTCATGTGACTGGATTAGAAG ATTCATAATTGTTAGAATGTCAGTGCTGCCCAAATTAGTCTATAAATTCAATGACATCCC CAAAAAAATCCCAGCAGCAGCTGTTTTATAGAAATTAGCTGAACAGATTTCATTTGCATG GATGAAGTGAAGTTTATTTAACTATTTTCCTATTGATAGACATTCACATTTATCATAATT AACATAATCCTCATATTTTGCCACCACATATTAATTTTTTGGTGAGTATCCTTACACATG TATCTTTGTACACATAATATGAGTAAAATTAAGGATGAATTTCTAGAAGTAGAATTGCCA GGTCASSGGGGTGTACATTTTAATTTTTGAGATATTGCCAAATCGTCTTCCAAAGTTGGA TCCTTTCCATTCCCAGTAATTATCAAATCCATTCTTTAGGAGATTTTGTATTAACTGAAC TGTTGACATAATTCCCTTTATATGCACACTATGAAAAGTATAACATATACAAATCTACCT ATATGGTCACAATCCTGGGAATTTATGTGACAGGTATTGGATGATGTTCAGCATCCTTGG CCTCGTGGAAATAATCAGACTAGATTTTCTTTTTGAAATGCAAATTGATAATAGCCATTA GTTGTTTCCTATGTTGCTGAGAACCCAGAGGAAGATAATAAATGTCCTTGGGATAGACTG GGGCTCCAAAGATCATGCTGTATACTTTTGAAAAAGCATTAAATTGTAGCCAGTGAGGAA TCAGAAACCAATCTTATTGCCATGTTCTTAACACAATCTTTTTCCTTATCTTCCCTCCCT TCCATTATTCCTTGCTGAAACATTAGCTTTGCACAATTACCTGCTGATAATGGTAGCTCT ATGTCTTTCTGCGTGCCTGCCTATCTGTCTGCTTGTGGAAAGATAAAGATAGATATTCCA TTTTCTAATCCCCTCTTTTGCTTGGGAAGAAACTAGAGGTTCTTCCTCAGATTGGGCACC AGAAGGTAAAGAGAGCAGGGGGTCAAAAATTGTTAAGAAGGGCAAGACTAGAACTTTCCT ACTCTCCCCATGCCCTGGTTTCAGAGAATCATTGAAGGGGCTCTTTGATGACCCTAGGAC ACTCACCTCTAAGCATAACAATATTCTGGAAAACCTTTGACAAATTATAGCAATTTGGTT GTAGAGAAGATAAATGTTTTCTCACCTGGCAAAGAACATGTGAAATGAAGCTGAATCTAC TGCAAACGGGGAAGTGGGTGTTTAACACTGCATGCATAAAGTATTGATTTGTATCTTTTC AAATTTATGGAACTGTTATCATTTTATATGTGTGCTTTATCTGATGTTTTAAAATTATGT TTTTGGGATTTGCCCAAATGGATACATGTAGACCTAATTCATTCATTTTTTATTGCCATA TAGTATTCCATAAAGATATTATCCTGTGTTTTCTCCTCAAAGTTACAGTTTTCCTTTCTA CATTTAGGTATTTCAATCTCCTAGAATTTATTTTTGTATGTGTGTGGTATGAGATAGGCT TCCAATATTTTTCCATATGGATTATTCCAACATTTTTTTCAATACATCATTTCCCAAGTG GTTCCTACCACTGCTTCTGCCATGCACCAGATTCCCATGTCTACTGAATCTGTTTCTCCC AGTAATCTCATTGTCACATCCTGAGTCAACACCACGTGGATTTAAATGCTCTATAGTAAG ATTTGACCTGGTCGATTCACATGCCCCTCCTTATTTGTCTTCTTGAAAATTATTTTGGTT CTTGTTTCTTTACTTTTCCATATCAGTTTAAGGGTCACATTTTCCAGCTCTTTTCTTACA ACTCTGTGGAAACTTTGTTTGGTAATGCACTGAATTGATTTATCACCACAGGAAGAATCG CCACATTTACAATACGAAGCCTTCCCACACATCAGTATGGCAGATCTCTTAATTTATACA GGTCTTCTTTAATATTTTTCTACATTTTATAATGTCTTTTTCCTAGGTACATTGCAATTT TGGTTGCCCTATTGAATACTTATTATTGCTAAACTGTCTTAATATTATTAATTTCTATAT AGTGATTTTGCATCCAGCAACCCTGCTGAGCTCCTTAAAGTTAAATTTTTCTGTAGATTC CCTTGGACTTCCTATTTAGACAGTTATATTATCTGCAAATAATGACAAACTTGTTTCTTC CTTTATGATTCTTATACCACTTTTTTATTTTTCCTGTCTTATTGAATTGGCCGGGACCTT CAACAAGATGGTAATTCAAAGCTATGACAGTGAGCATATTTATTTTGCTCTTGATTTTAA AGTGTGTGCTTCTAATGTTTCACTATTATGATGTTTATGAGAGTCTCATAATTACTTTTT ATATCAATTTTTTCCATTTCTTGTTTGTTGAGACTTTCATCATGAATGGGTGTTGGCTTC TATCAGTATTTTTATATTTATTGAGGTTATCATATAGTCTTTCATCTGTTAGTATAGTAA ATCTGTAAACTAACAGGTTTGTTAGTCTATTTTTAATGGTTCCAGCTGATCATTTTGCTG GATTCAATTAGGTGTATTTCATTTTATCTATTTTGCTAAATGAGGCTGGCCTGTAATCTT CTTTCATAAACTGTCCCTTTGGGATGTTCCAACTCTCTCCTCAATTGTCCCTGGGAACTC CAACTTATTCTTACTTTGTTTAGCAGAGCAATTCATCTCTCTCCATCTCCTACAATGTTT CTCAACAAGTGATCTAAGGATCTCTATGCACATATAATTTTAAAATCTAACATGTAAGAT TAATTATTATATTCATAGAATTTCTAACTTTTTATGTTTTCAAAAACAGTGTATGTGCTT CAAAAACTGATTTCATTCATAATGGTTAACATGGAGTTAATCATAGGCAGTTTATCTTGA CTAATGTATTTTATTGTCACCTGACAGTTATTTACACTTGTCAGACTTGCATTTGCCATT ATTACTGTCGCTTTGCATAATATCACAATTCAAAAAAAAGAGTCATCACTTCGAAAGTGG TTTTCTGAAGCATAACAATAATGATAAAAATAGAAATATAAATCCTACAATGTGAAATAA GGTCACAATGTAAGTAATGAATGGCCTCCATTGTAAAAATACAAAGCATGAATCTGTTTG TCATGAGACAAGTGGATAATGGATGTGGATAATCTCCCAAAACCAGACAAAAATATATAA GAAAATGGGAGCCATGACAATGAATAATGATAATTGAATTTTATTGGACCAATGATCCTT TTGTCCTAGTACCCAATTTATTATCTGCTAACGTTTTTGTCAAGTAATGATATAAAGCTA TCAAAACTTCCATATTATTTTCCAATAAAGTTTATCAACCCCACTTTTAAACCAATTGAA TATCTCTACTACAAGCACAAAATAAGGCTTTCCCAGTATAAAATTGTGAAAATTTTATGT TAAAGGTTGAGAAGACAACAAAACTATAGAGTCATCATTCATCCTCATATCAAAAATAAG CACAATTGCACTCCTGCTGGTAAGATACAAAACCAGTTTCCAAATCAATTACAAATATTG TGGTTGGAGAAGAAACAAGAAAAACAATTGGTATAATTCCTTAATTAGAAGTATTGCATT CCTTATAATGTGACTGGCATTTAAAGTCAATGACTGTTAGGTATACGAGCAGACATTTTA TTTTATAGTTGAACAAAACTATGAATAAGTAAGTTGTCAGCATATCTTCACTATATATAT GGGAAAGACTCAGTGACTTTTTGTTCTATTTATCATGAAGCTAATTTTAAAAGAAGAGCT CTCTGGTTTAATTAAAAATTAGTTTGAACCTATGAAACAGACTTTAAAAGGTATGTTGGC TTATGCACTAATGTTTGTAGCAGCGTTATTCACAATAGCCAAATATGTCCATCAACAGAT GAATGGATAAAAAATGTAGTATATATGTAAACAATTGAATATCATTCAGTTTTAAAAATG AATGAGATTCTGATACATGCCACAACATTGACAAAGCATTGTGCTAAGTAAAATAACTCA AACGCAAAAGAACAAATGTTGCATGATTCCACCTTTAGTAGGTACCTAGAACAGGCAAAT TCATGGAGACAGAAAGTAGAATGGAAGTTGCCAGAGAGGGGGAATGGGGAGTTATTATTT AATGGGTACATTTCTGTTCGGGATTATAAAAAAGTTTGGAAACAGATAGTGGTGATAGTT CCACAATACTGAATGTACTTAATGCCACTAAATTGTGTACTTAAAAATGGTGAAAGTGGT AAATTTTATGTTATGTATATTATGCATATTTTACCAAAACTTTTTTTTAAAAGAGGAAAG GTGTTAAATTGCCATTGAAGAAACTCCAGCCACATGGTCAGCAAGGAAAGGTATCACTAT CCAGATAGAAGATGCTGCTCCCAGATCCCAATGCTAGTGCATACCTTATTCACGGAACGC AGCTGGGGGTCTGTAATATGCCTCCAGATTCTGATTTGCTGTTGAGGTACACAGTGGAAA TTGTGAATACAAACTGATTGTGGTCACTTAGTGTACATCTCTTTGGCCTACTGGGCTGAA ATGAAAAGTGAGTAAGAGAGGCTGCTCTCCCACACTGAATTGCTTTGGCTGGCAAATGGG AAGGGCTTACACAGAGTTTTAAAAATAAGGAATGAAATGTTTTAAATTACCATTTTTATG AATACAAGTTGGGTATCTCAGTGATCCATTCAGAATTTGAATATTATGAATCTTTGTTTT CAGAGCCTGAATATCGGGATTTGTAACAATCAGACATCAGTGGTGCAAGTGGCTCAACGG CTGAGAACTTGACTTTTTCCCAATACCCAATGATTTTCTTCATTCACCAGAAAAAGCACT TAGGAAATAACACATTGGGCTTATTAAAAATTACATCTGAATGCTGCAACCCAACATCAA GAAATACCTTGCAGAGCCAAATACCACCAAAGAGAGGATGCAACTCCATTAGCTGCTGTT GAGCTGGGGCACTTGCCTTCTGGTGAAGCACTGAAAATAGTCTTTAGAGAGCACTCTCTC CATAATTTCTGGGTCTGTGTTTGATTTGACAAACCAGAAATATCCATCAAAGCTTCCCAA CAACCTATAATTCTGAGTTAAGATTTTCTAATTTAGAAAACATAAAGAGTGTGGGGGGGA AACCAACAAGATATGGAAGCTGACAGCTCAAGCAGCTATGCAACCAGATATAGATTCCTA AATGATTTCAAACACGATTTCTCCTCTCACAGATGTGAAAAAAAATTATTCACTTTTTAA CTTGACTTTTACTTTTATTTAAGACATATATGTTCATAATTTAAAAGGTCACAGAGTTTT ATAAAGATTCTGACAAAAAAAACAGCAATTTCATTTTTCTCATCTCTTCTTTCCAGAGGC CACCACTTTTAGCTTTTTTTACCTGTGTCTTCTGATACCTCCCTATCTCCAATAATATAC TCATAATACTAATTTTTAATTTTTCAATTTCTGATGTTATTTATTGGCTTCCTATTATGA GAAAAAACTTAAGGCCACATCCACAAACTCATTTCCTCTCACCCTCATCTTCCTGTTACA GTTATATTGTAATCTTCAGTTAGATCATCAGATAATATATCCATTGTATGACTACATATA TAATTACATTACATTGCTAAGTAAACACTGTTTACAGCTGTGTCATGCAGTGTACTTTGA CTATATTTCCTTTTTGCACAATGTTTTTGCTTTCCTTGGAGTTAATAAGGATTTTATTTT TTTGTTTGCTCAGTTTTCTATTTATCCCTAATTTGTTCCCAAACTCTTCTCCAGATGTTT AATTGTTGCTTCAATTTATTTGCACACATCAGGTGCATGTGTATCATTTTTTTCCTATGG GAATCACAGCTAGAGTACTTCATCTTCTGCCTCATTCTGACCTAGATGTTTTCTAGGCCT GATGCACAGCTGTTTTCTAGAGATTTTTTCTTCTTCATTCTTTTTTTGGATTCCTTTTGC CTCTTCCCTGGATCGCATATATGTTTCTTGTTTGAATGGAGCATATCCTCTAGTAACATT TTTTTTTAAGACAAAGTCTCTCTATGCTGCCCAGGCTGTTGGCCTCAAACTCCTGGGTTC AAGTGATCCTCCTGCCTCAACCTCCCAAGTAGTTAGGACTACAGGTACATACCACCATGC CCAGCTTCTAGTAACTTTATTAGAAGGGTAACTAAGAAGTACATTTCCCAAGACTTCATA TATTCTACCTTCTTTGACAGTTTGGCTAGGGAAAGACTTCTGTATTAGAAATTATTTCCC TCTTAATTTTGAAGGCCTTGCTCATTGTCTTCTAATTTCTTTCTAGACCTCCTATTAGTC AAACAATATACCTCCTAGTCTGATTCTCTAATTTTCAAACTTTTTATCACTTCCTATCCA TTTCTGCTTTTTTGTTCCACTTCCTGTGAGATTTCCAAAACTTTATTTACAAGTCCGCCT ATTAGTGTTTTACTTTTGCTATCATATTTTTGTTTTCAAAATTCCATTTTGTCTTTGAAG GTACTTTTTACATCCTTCTGCTCTTGTTCTATACGTGGAGCCTCTTCTCACCTCTTAACT CTCTAAAAATATTAGTACAGGTTTTTTTTTTTTTGTTTTTGTTTTTGTTGTTGTTGTTGT TGCTGTTGTTGTTGTTGTTTTGGAGACGAAGTCTTGCTCTGTCACCCAGACTGGAGTGCA GTGACGCGATCTAGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCCGCCT CAGCCTCCGGAGTAGCTGGAACTACAGGTGCCCGCCAAGGCGCCCGGCTAATTTTTTGTA TTTTTAGTAGAGACGGGGTTTCACCGCGTTAGCCAGGATGGTCTCGATCTCCTGACCTCG TAATCTGCCCGCCTCGGCCTCCCAAAGTGCTGCGATTACAGGCGTGAGCCACAGCGCCTG GCCAATACAGTTTTATTTCTCTAACTCTTCTTCTGCCCTCTTTATTGTCTCTTTTTCTTT TCTTTTCTTTTCTTTTTTTTTTTTTTTTCAGTTGTTTGGTTGCTTTGGTTTCTGGTGTTA GAAGCATCCTTCAAATTCCTGATGCTCCTTGGCTGGCTGTTCATGTTAAAAGTGCAGCAT CTTCATGGGCTCTAAACCAGATTATCTTCTGGAAACTTAACGAGGAGCAGAAGTGAATAG GGAGGGGAGTTGTTTGCCTTTCAGTAGGTAAACTTTTCTTTATATGCATTTTTCAGTATG CAACTGTTTCCCTCTTCTTCAACTCTTAAGAAAATGAGCATCCAATCCATTTCTGGAGCA GAAACAGTTTCTCATTGAATGGATGGCATAAAGCAGCATCTAGAAAGCTCAACTGGTTCC TGTAAAAAACTTTCAAGCAATTCTTTCAGCCCTACTTATCACCCTCCCCCAAACCTTGCT TTCAGAGATACTTAGCATCTCTAAGTTCCAAGCAGTTCTGGGGCTCTGCAGCATAGCTTG GCTTGCTTCTGGGTATTCCCTGCTTCAGCCAGTTTTCAGCTTTCTTAGGTCTGCTCTGTC AGTTACCACTCATCAGTCTGCACTCCAGTTTCTAAAAACTGGGAGTTTTTAGATGAGCCT CCAGCTTCTTGCAGGATAGGGAGGCCCTGGCAGACATAAACAGGAGAATCCTTGCTCTTC ACCCGCTACTGCCCTCTGCCTAACCCCAGAACAGCAGAAGCTGCTGGCTGTCCCAGCCAC ACACATCCCTCCCCACTCCATCTATTTGTGTGGCCCTTCTTTTTCCCTCAGTCTGTGTCT TGTTTCCTCTCCTTACCTTCTTCCCCCTCATCACCTTGGTGATATTCTTTGCATGTTTTT TCTTGGAGCAGTCAGAGCAGGTCATCCCTCCCTCCTGGAAGAAGGAACCCCCTGAGTACA CTCCATGGCACAGTTGACACTCCTTTTCTACCCTCCTTCCCAGATATGTCTTCACTGTCA TTTACACCTTTGAAGCCTTGATAAAGATACTGGCAAGAGGATTTTGTCTAAATGAGTTCA CGTACCTGAGAGATCCTTGGAACTGGCTGGATTTTAGCGTCATTACCCTGGCGTGAGTAT ATCTCTATGCAGATCTGCATGTGTGATAGGAGAGGGGACAAAATGGTGGGTAGGAGACAG GGCTAACATGCAGGTCCCACTTGGACAGAGTGTGGAGACTCACACCATGAACGTTTGCTC CAAGAACCACTGCAGGAATATACCAGGAGAACTGAAAGAATTCATAGATCCTTTTAAATA AGTGGCACACTGCTGGAAATTCTGCAAGACAGGTGAAAAACTGTGAGCTCCCAAAGTGTG AGGGGGGGAAACCTGCCTCCAAACATACATCCCAACTGGGTAATCTGAAAATCCAGATCA CAGGAGAAGAATTTACCCTTACCTGGAATGTAAATAAATTTAGGAAGCCACACAAAATAT AAAAGTAGAAACAGCAAAGGGAAGTGCCTTGTAGGCACTCCCAGTCTCCAGCTTGAGACC AGGGAATCCATCCCTGACTATGTCTCACAGGCACCCTTGGGGAAGGCAGCCAGTGGAATC AGGGAGGGGTCACAGGATGAAAGAAGTTTCAACTAAAATTAGTAATAATTTCAACTGGGC ACAAATTTTCTTGAGCAGAATCCAGGGGGCAAATGGGAACTGCAAGCAGACAGTGAGGGA TGCGGTCCAAAAGTCATGCTTTCTTTCTCAGTGGGGAAGCTCACAGCCTGGGGCAAGGTC TGAGTAGGGCACTGCAGGAGCAAGACCAGCCTCACCAACTACATGAGAGCTAGGTGAGGC CTCTCACTAGCAGCTATCCCCCACTTCCCTGGAGAACTCTATGGCACAGCAGAGGCAGCC AAAATTCCCTCTGGAACACAACCCCACTGGCCTGAGAACCAACCCCCATCCACCACAGTG GCCACAGCAATCCCTACCCAAGAAGGGTCTGAGTCTAGACTCACCTACCCCTGCCCCCAC CTGATGGTATTTCCCTACCTGCCTTGGTAGCTGAACACAAAACCCAGAAACTCTCGGAAG CTTTATGGCCCTGCTCATCACCTAAGAAACTAAAATATTTACCCTGGCCAACTTAGGGTG AGCTTAGATCCCCCTTCTACTAATGCACTAGTGCTCTCTTGAAAGCATCACCTACCGGCT GGAGGTCAAACAGCTCAGGCCATTAAAGCAACTCATGACAGAATAACTTTGATCCCAGGA AAAAGAAAACAACAACCAGTTGCACTGCCTGCAACATCCTGGCTAACCAGACGTCCTGAG TCTGTCCATATGACAACTTCACTGCTAACATAACTAGCATTCAAGAAAGCCAGCACACTA AATCTATCTACAGCCTAGGACTCTCACAGACTCTACTTCATTCCTCTGCCACCTTCACCA GAACAGATGCTGATATCCATGGCTGGGAGACTTCAAGATCAATCACATCACAGGACTCTT GCAGGCATTTCCCAGTACTACCCCAGAGCCTGGTAGCCCCTCTGTGTGGCTAGGCCCAGA AGAACAATAACGATCACTGCAGTCCAGCTCTCAGGAAGCCCCATCCCCAGGGGATTGGGG AGAGCACCACATCAAGAGATCACCCATGAAACAAAAGAATCTGCACAACAGCCATTCAGT TCCAGATCTTTTCTCTGAAATAGTCTACTCAAATGAGAAGGAACCAGAAAAGCAATTCTG GTAATATGACAAAAGAGGGTTCAATAACACCCCTCAAAAGATCAAACTAGCTCCCCAACA ATGGATCCAAACTAAGAAGAAATCTCTGAATTGCCAGATAAAGAATTCAGGGCTGGGCAC AGTAGCTCACGCCTGTAATCTCAGCACTTTGGGAGGCCAAGGTGGGCAGATCACCTGAGG TCAGAAGTTCAAGACCAGCCTGAAGCCAACATGGAGAAACCCCGTCTCTACTAAAAAATA CAAAATTAGCTAGATGTGGTGGTGCATGCCTGTAATCCCAGCTACTCAGGAGACTGAGGC AGGAGAATTGCTTGAACCTGGGAGGCAGAGGTTGCGGTGAGCTGAGATCGCGCCATTGCA CTCCAGCCTGGGCAACAAGAGTGAAAATCCATCTCAAAAAAAAAAAAAAAAGAATTCAGA AGGCTGATTATTAAGTGATTCAAGGAGACACCAGAGAAAGATAAAAACCAACTTAAAGAA ATTTTTAAAACAACAAGATATGGATGGAAAATTCTCCAGAGAAATAGATATAAAGAAAAA ACAATCACAACTTCTGGAAATGGAAGACTCACTTAGAGAAATACAAAATGCAGTGGAAAG TTTCAACAATAGACTAGAACAAGTAGAAGAAAGAACTTCAGAGCTCGAAGACAAAGCTTT TAATAGACCCAAACTAAGACACACAAAAAGAAAGAATTTTTAAAAAAATGACAAATCGGC CAATAAATTTGGAATCATGTGAAACAACCAAACCTAAGAATAATGGGTGTTCCCAAGGAA GAATAGAAATCTAAAAGTTTGGAAAACTTATTTGAGGGAATAATTGAGGAAAACTTCCCT GGCCTTGTTAGAAATATAGATATCCAAATACAAGAAGCTCAAAGAATACCTCTGAAATGT GTCACAAAAAAGATCATCACCTAGGCAAATAGACATCAGGTTATCTAAAGTCAAGACAAA GAAAAGAATCTTAAGAGCTGTGAGACAAAAGCCTCAGATAACCTATAAAGGAAAGGTTAT CTGATTTACATAAGATTAACAGCAGATTTCTCAACTGAAACCTTGCAAGCCAGAAGGGAT TAGGGTCCCATCTTTAGCCTCCTGAAACAAAATAATTGCCAGCCAAGAATATTGTATGAA GAGAAACTAAGCTTCATAAGTGAAGACAAGATAAAGTTTTTTTCAGACAAACAAATGGTG AGAGAATTTTCCACTACCAAGCCAGCACTACAAGAAATGCTAAAAGGAATTCTAAATCTT GAAATAAAACCTTAAAATACACCAAAATAGAACCTCCTTAAAGCATAAATATCAAAGGAC CTATAAAACAATAACACAATAAAAAAAGGAATTAAGGCAATAACTAGCATAATGAATAGA AGAGTACCTCACATCTCAATACTGATGTTGAGTGTAAATGCCCTAAATGATCCACTTAAA ATATACAGAAGGGCAGAAAGGAAAAAAAAAATCCACCAACCAAGTATCTACTGTCTTCAA GAGACTCACCTAACACATAAGGAGCCACATAAACTTAAGGTAAAGAGATGGGAAAAGATA TTCCATGCAAATGGAAACCAAAAGCAAGCAGGAGTAGCTATTCTTATATCAGAGAAAACA GACCTTAAAGCAACAACAGTTAAAAAAAGACAAAGAGGGACATATATAATGATAAAATAA TTGGTCTAATGGAAAAATATCATAATCCTAAATACATATGCACCTAACACTGGAGCTCCC AAATTTATAAAACAATTACTACTAGACCTAAGAAATGAAATAGACAGCAATATAATAATA GTGGGGGATCAATATTCCACTGACAGCACTAGACAGGTCATCAAGACAGAAAGTCAACAA AGAAACAATGAACTTAAACTGTAACCTAGAACAAATGGACTTAACAGATATTTAAAAAAC ATTCTACCAAACAACTGCAGAATATACATTCTTTTCATCAGCACATGGAATATTCTCTAA GATAGACCACGTGATAGATAACATAACAAATCTCAATAAATTTAAGAAAATAGAAATTAT GTCAGGTATCCTCTCAGAGCACAGTCGAATAAACCTCGGAGTTAACTCCAAAAGAAACCC TCAAAACTATACAAATACATGAAAACTAAACAATCTGCTCTTGAATGATCTTTGGGTCAA CAATGAAATCAAGATGGAAGTTTAAAAATTTTTTGAACTGAATGATAATAGTCACACAAC TTATCAAAACCTCTGGGATACAGCTAAAGCAGTGCTAAGAGGAAAGCTCATAGCATTAAA TACCTACATCAAAAAGTATGAAAGAGGACAAATAGATAATCTAAGGTCACACCTCAAGGA ATTAGAGAAACAAGAACAAAACAGACCCAAACCAAGCAGAAGAAAAGGAATAACAAAGAT CAGGGCAGTACTAAATGAAATTCAAATAAAAATAATAATAATACAAAAGATAAGTGAAAC AAAAAGCGGGTTCTTTAAAAAGATATAGAAAATTGATTGACCATTGGCAAGATTAACCAA GAAAAGAAGAGAGAAGATCCAAATAAGCTCAATTAGAAATAAAATGGGAGATATTACAAC TGATACCACAGAAATACAAAAAAAAAATCATTCAAGGCTACTATGAACACCTTTATTTGC ACAAACTAGAAAATTTAGAGGAGATGAATAAATTCCTGGAAATATACAACTGTCTTAGAT TAAATCAGAAAGTAATAGAAACTCTAAACAGACCAATAACAAGTAGCGAGATTGAAACAG TAATTCAAAAATTGCCAAGGAAAAAAAGTCCAGGAGCAGACTGGATTCACAGCTGAATTC TATCAGACATTCAAAGAAGAATTGGTACCAATTTTATTGAAACTATTCCAAAAGATGCAG AAAGAGGGAATCCTCCTAAATCATTTTATGAAGCCAGTATCACCCTAATACCAAAACCAG AAAAGGACTTTTAAAAAAGAACTACAGACAATATCCCTGATGAACATAGATGCAAAAATC CTCAACAAAATGCTAGCTAATTGAATCCAACACATATCCAAAAGATAACACACCATGATC AAGGGGGTTTCATACCAGGGAGGTAGGGATGATTTAACATACACAAGTCAATAAATGCTG CACGTCACATAAACAGAATTAAAAACAAAAATCATATGATCATCTCAATAGGTGCAGGAA AAGCATTTGACAAAATCCAGCATCCCATTATGATTAAAACCCTCAACCAAATTGGCATAG AAGGGACATACTCCAAGGTAATAAAAGCCATCTATGACAAATCCACAGCCAACATTATAC CAAACAGGGAAAAGTTGAAAGCATTCCCCCTGAGAACTGAAACAAGACAAGGATGCCCAC TTTCACCATTTCTATCCAACATAGTACTGGAAGTCCTAGCCAGAGCAATCAGACAAGAGA AATAAATAAAGGGAATCCAAATCAGTAAAGGGGAAGTCAAACTGTTGCTGTTCACCAATG ATATGATCGTATGCCTAGGAAACCCTAAAGACTCATCCAAAAAGCTCCTAGATCTAATAA ATGAATTCAGTAAAGTTTCAGGATACAAAAACAATGTATACAAATCAGCAGCACTACTGT ACACCACCAGAAACCAAGCTGAGGAACAAATCAAGAACTCAATCCCTTTTACAACAGCTG CAAAAAAAAAACAAAAATAAAAACAAAAAAATCTTAGGAATATACCTAACCAAAAAGGTG AAAGATTTCTACAAGGAAAACTACAAAACACTGCTAAAATAAATCACAGATGACACAAAC AAATGGGAACACATCCCATGCTCATGGATGGATAAAATCAATATTGTGAAAATGACCTTA CTGCCAAAAGCAATATACAGATTCAATACAATTTCCATTGAAGTACCACATCATTCTTCA CAGAAATAGAAATAACAACCCTAAAATTCATAGGGAACCAAAAAGGAATCCACATAGCCA AAGCAAGACTAAGCAAGAATAACAAATCTGGAGGCATCACCTTAGCTGATTTCAAACTAT ACTACAAGGCTATAGTATAGCCTGTACCAAAACAGCATGATACTGGTATAAAAATAAGCA CATAGACCAATGGAAAAGAATAGAGAACTCAGAAATAAAGCCAAATACTTACAAGCAACT GATCTTTGAAAAAGCAAACAAAAACGTAAGTGGGGAAAGGACACCCTATTCAACAAATGG TGCTGGGATAATTAGCAAGCCACATGTGGAAGAAAGAAACTGAATCTTCATCTCTCACCT TATACAAAAATCAATTCAAGATGGATAAAAGGCTTAAATCTAAGACCTGAAATTATAAAA AATTCTAGAAGATAACATTGGAAAAACTCTTCTAGACATTGGCTTAGGCAAAACGTTCCT GACCAAGGACCCAAAGCAAATCCAACAAAAACAAAAATAAATAGATGGGATCTAATTAAA CTAAAAAGCTGTTGCACAGCAAGAGAAATAATCATCCAAGTAAACAGACAACCCATAGGA TGAGAGAAAAATCTTTGTTATCTATGCTTCCAGCAAAAGACTAATATCCAGCATCTATAA GAAACACAAATAAATCAGCAAGAAAACAAACAAATAATCCCATCCAAAAGTGGGCAAAGG ACAAGAATACGCAATTCTCAAAAGAAGATATATAAATGGCCAATGAACAGGAAAAAAAAA TGCTCAACATCATTAATTATCAGGGAAATGCAAATTAAAACCACAATGAGATACCACCTT ACTCCTGCAAGAATGGCCATAACCAAAAAATAATAGATGTTGGCATGGATGTAATGAAAA GGAAACAATTTTACACTGCTGGTGGGAATGTAAACTAGTACAACCACTAAGGAAAACGTT ATGGAGATTCCTTAAAGAACTAAAAGTAGAACTACCATTTGGTCCAGCAATCCCACTACT GGGTATCTACCCAGAGGAAAATAAGCCATTATTAAAAAGACACTTGCATATGCATGCTTA TAGCAGCACAGTTTACAACTGCAAAAATATGGAACCAGCCTAAATGCCCATCAACCAACA AGTGCATAAAGAAAATATGGTGTATGCATACCATGGAATACTACTCAGTTGTAAAAAGGA ATGAAATAATGGCATTGGCAGCAAGCTGGATGGAGTTGGAGACTATTATTCTAAATGAAG TAACTCAGGAATAGAAAGTCAAGCATCATGTGTTCTTAATTACAAGCAGGAGCTAAGCTG TGAGGATGCAAAGGCATAACAATGACATAATGGACTTTGGGGACTTGGGAGGAAAGGTGA GACGAAGGTAAGGGATAAGAGACTACACACTGGGTACAGTGTACACTGCTCGGGTGATGG TGCACCAAAATCTCAGAAATCAGCGTTAAAGAATTTTTCTGTGCAACCCAACACCACCTG TTTCCCAAAACTATTGAAATACAAATAAAGAACTTATTCATGTAACCAAAAACCACCTGT TCCCTCAAAACTATTGAAATAAAATAAAATAAAAAGATTTGCATGGGTGTCCATGTGGGG CTTATGGGTGTATCCAGGCTCACACTCTGAAGAAGGTTCTTCATCTCAGTTCAGCTTCCT CATTTGGAATTGTATGGCCTGGGGTTCTTGCCACAGAGTACCGCTTCGGGGGTTGCTTTC CAAGATTAGTCCCATGCCCATGAGGAGATGTCAGAGACCTCAAATCTGAGATAACAGTTA GCCTGTCACAGTGGATCAGAAAAGGCCCAGCCTTGTACCCCCCTGACATGGCGGTCTCCT TCTGAGAAATCAGTGTCTGAGGAGACTGGTTGGTCAAACTCCTGTGTTTGAAGATCCGTC CACATGCAGTGTTGCCTAGATCACAATGCATCTTCTCAAAGGGTCCACTCTGAAAGATGT TGCAATAATGAAATCAGTGTCAGTGTCAGTCTATAGATGGCAGTGTCACTAGATTCTAAG TGTCCATTCTGCATCCTTTCTAGGCTAATGATACCCCAGGTTATCCTTATTTATGCTGCA TGCATCCCCAGGACCCTTGCTTGCAGAGATGCTGACTAACTTCTGGTCCTACAGATATGT TGGCACAGCAATAGATCTCCGTGGGATCTCAGGCCTGCGGACATTCAGAGTTCTTAGAGC ATTAAAAACAGTTTCTGTGATCCCAGGTGAGCATTTATTCACAGAGCATATGTTGGTTTT CACAGCCTAACTCCCTTCTCTAATTTTCACAAGAACCCTGTAAGGTTCCAGGGCAAAGAC TATTGTCCCCACTTACAAGACAAAGGAACTGAGGCCTGGAGTATAGGAAACATAAACCCA AGGCCTCTTCACTAGTAAGAGACAGTGTGACCTGGAAGCCTATTGTCCCAACTCCTAGGC CAGTGCTCTTCCCACATACCACATGGCCTCACAATCAATACGTAACAGGAGGGGCATGTC TTCACTTTCTGTTCTGGTTGTGGGATTCAGGCAAGACCCCTCAGTTGAATTTTTATTTAT ACCTTAAACTAAAGGATTAAACTTGGAAATGGGGAGGAAGGGAGAAAGCAGTCCCAGTAT CTTCACTGTTTCCGTAGGTCTAAAATCCTGTCTTTAGACACCCAGAGGAAAAAAGAATTA GGCAATTCTTGATTACTGAGAATTCAGAAACAAAAGTAGTCCATCAAAGCTCATTGTGTG GGGCCTCTAAGATCTCCACTGTCACCAGTAGCCCAGCGACTACTCTTCAGCCCCCATGAG GTTGATGGTGAGGATGGGGAGGCCAGAGACAAGGCAAACTTTCTGAAATGGGACTTGATG CTTCCCCTCTGTGGCCTGATAGGTTCTCTCCTCTCACCCCAAGAATTCGTTCTCTTATCT GTAAACAGAAACACCCATTCTAGAGTGGAAGTCTTTTCTACCATTTCTTGTCTGGCTCTG CAGGGAAAGATAGGAAACTCTCTGCCAATTCCCACTGGACACACCTCTTGAAGAGAGCCC TGGTCCTGCCCCTTCATATCTCTCTCACTTACTCTCAACTGGGAAGACCCTGAGTCTTAT TTGCAAGACCTGGTCTGTCCATACTGTCTCTTCTGCCTCATCTCTCACCTCTCCTCACAT CTCACCATGCTCTGGAATCCACCTACGGTTCTGCAAATACTCCACACCATTGCTCACCTC CCAGCCTTCTACTGTGCTGCCCCTTCTTCTGGGATATCCTTCCTCCTCTCTTTACTCAAC TCACACTCATTCCTCAGGGCTCTGCCAAGGTTTTACCTCCTCCCTGGTTTTCCCACCTTC CTCACACTCCACCCACCCCATTCCCACCTCTGGAACTTCAGTTAAGGGCTGCTCCCCTGT GTGCCCATCTGCTCAGTAATGCTACAGACTTTCCCCTCAATTGTCAGACAACGGCACTCG ACATGCTGCTGCCTTAGCCATCACTCAGCTCCCAGTCCAGGGACTGACACATGTAGTGCT CAGTGGATTATTTGCTGACCAAAATGACTAACCAAAAATAAATCCTAGATTGCTTCCCTG GTCAGAGCTCTTTCTGCTACCTGCGAAGCTGCTCTCCCAAAGAAGTTGATTTTACTTACC AGAGCAACAAATCATCCCTCCTTAAACAAGCAGGTGTATCCTTCTTTGCTGCCAGCTGTG CTTGGCACAAGTTCTATTTATGCCCCAGTGACTGTCTCAGTGTTTCTTATAGTCTCTTTC TATTGCTAAACTCACCTCTCTGTTTCTCTGTGTCTCTTTCTTTTTCTTTCTCTTGGTCTA TTTTTGTTTCACTCTATCTCTCTTGCATCTCTCTCTCTCTCTCTTTCTCTCTCTCTCTCT CTCTCTCTCTCTCACACACACACACACACACACACTCACACACAGTGAGGTTCATGTGTC CCAAGTCTCAAGCTGCCTCCCAGATGCATGCAGAAAGACAGATGCCAAATTACTGGGGTG AAGTTAGTAGCTTCATGCCTAGCACAGTGCCTGGAAAAAGTAGGTACTCAGCATAAATTA TTTAAATTGAATTGCTAGTGTCAGGAATTTTATTAGCCTCAAGATGATAAAAAATGCATT GCCTGGGTCTCAGTTGCATCGTCTTTATAATGAGAATAGCCCCTGACTCTCTGCCTGCCT CTCAGAGTAAATGCAAGCTTCGGATGAGAAAGGTCTTGGAACTGCCCTGGAGTGTGTACA TACTTCACTCCTGTATGTAGAGATGAAGGAGGATGGCTAAGATGAAGAAGTGTGCTCCGT GTGTGCTACCTCATCCATGTGGTCATACCACTGTCATCTTCCCGCAGGCCTGAAGGTCAT TGTGGGGGCCCTGATTCACTCAGTGAAGAAACTGGCTGATGTGACCATCCTCACCATCTT CTGCCTAAGTGTTTTTGCCTTGGTGGGGCTGCAACTCTTCAAGGGCAACCTCAAAAATAA ATGTGTCAAGAATGACATGGCTGTCAATGAGACAACCAACTACTCATCTCACAGAAAACG TGAGTGGAGTCTCTTCCATAGGGACCAAGGTCTGGTTGGACCCTTGGTATCATATAGGGA CAGGGGATATGGAGCCCCTGTGTGTGTGTATAGACATATATTTTGACTCCCCTTTCCTGC CAAAATGGTTCTCTTGAGTTGTGTTTTACTTTTGGGTATTCTATTTCCTGTTGCTCAGCA TCCCTCTTGCAAGGAAATGAGCCATAAATCTAGAGGCAAGTCATTCTACCTCTGCTGGGG GAGATTGAGAGCCGAAGTCACTGCTTAGCTCTTCTCTTTTCAGATGTGACTTTACAACTT CCATGTGGCCCAGCATGCCCACTGAGTACATGTAGCCAAAGGAGCTCACTTTGGAAGACT TGGGAATATTGCATTCACCACACAAGGCTTGGGTTGGAGGGTTCAGAACCATCTGTGAGG CTTTCTTTTCTTCCTTTCTTCAGCAGATATCTACATAAATAAGCGAGGCACTTCTGACCC CTTACTGTGTGGCAATGGATCTGACTCAGGGTGAGTTCCCAACTTATGACAAAGCACGAG TGCCCAACAATTCCTTCTCCATCTTGTCCTTGGCATATTATGGGCACAGATGGGGTTGCA GGTCCTGGGAAAGATGTATAAATAAATAAATCATGGCCTTTGCCAACCCAGAGCTCATGG GGAGGGAATGTATGAGTTAGCTTTCGCTGTGTAACAAATGACCCCAAAATTTTGTGAATT AAAACAACAATGTATTTAATTCATGATTCTGTGCATCAGCATTTTGGACTGGGATCAGAT GGGCATTGGGTGACATTGACTAGGCTCATTCATGCATATGCAGACAGCATATTAACTTGG AGATGGCTGGTCTAGGACGTCCTGTCTCTGCTCATGTGGCCTCTCACCCCCAAACAGGTT AGCCTGGGCCTGTTCTCATGGCAGTGTTCCCAGAAAAACAGTGGAAGTTTGCAAGGCTTC TTCAGGCCTAGGCTTAGAACCAACACAATGTACCTTTTGTACATTCTGATACATTCTTGA CTAAAGCAAGTTGCAGTCCAGTAGAGATCCAAGAGACAGAGAAATAGACTTCATCACTCA ATGGAAAGAGCTGAAAAGTCACATTGGAAAGGGTGTGGATACATAGAGACAAGAATTATA GCCATTTTAATAAATAATCTACCCCAGGAAGGAACCAATACTTATTTGTGTCAAATGCTC TACACTTAATCATCACAGCTAACTCCATTCTATAGCCACATTACCCTCACAAGTAGAGGA AAAAACACATGTATCAGACTTTGCAATTTCGAATTCTTACAGAGGCCAGATTCATAACAA CAAATGAGTGGAGTGGGCCAGGAGGGAACCTAGGAGCTGAGAGTTCATGCCTCCTCTAAA GGGGACAGCCCCACTGCTCGGTCCAGCTTTTGGTTGCCATGTGGAAATGTGGGTTCACTG TTTCCAGCTCTTCCAACTTTTAAAATAGAGATAGGAAATTTAGATTTTTATATAAAATCT CCCAACCGTAAACATTGGCTACCTATTCCAAAAAATGTCTACATATTGTTGGGCCAAACA AAATATTTCTGTGGGCCAAATCCAGCCTATAAGCTGCTAGTTTGCAATTTCTCATTCATA GACATAAAGATGCTACCCTGTGGAAAACAAGTTCTTTACTAGAGGCTTCATAGGCTGGGC AAACACAGTGAGGTGGCGGTGTGAGGCAAGAGAGAAAGTAGGGAAGGCTTCCTGGAGGTG GTGTTTGAGGTGAGCTTTCAAGGGCAGGTAGGATGTCTCCAGGCTGGGTGAATACGAGAT AGTTTCAGCGAAGACCATTCCAGGTGGCAGAACAGAATGTGAGACAAAGCTAAGGGATGA GGACCCCTCAGCTCTGCCTGCTGCTTTGGCCACTGGAGGAAGGCATGAGCAGAAAGACTT TAGGCAAAAGATCAAAAAGGCCCATCTCACCCACCTCCATGGCTGAGAGCTTGGAAAAGC AGAGCTCACAACACCCCCAGTGCCTGAGAGGCTGACGGCTCCATGTTGCCCAGGTCGACC TGTGTGGAGCCTGCATGAGGCTCCACCTTCATCAGCTCACTAGGAAAACTATCTCAGTTG GAGCTGAGGACTGGAAACAAGCAGGAGCATTTGGTCAACATCAGGTTCCTTGGACTGTTC TTTCACAAATGCACTTGGCATTTGGTTCTTTGGCTCATGAGAGGGTTGTTCTGACATCAG TTTTCATTTGCCTTTGACTTAAATGTAAACCAAGAGCCCCTCAGCATCCTTCTGTCTAGA TAATGGCATTTCTCAAGGCAATGGCTCCAGAGCAGCTCAAGTCCTTTTTGTGGTAAAGTT GTTGGGTCCATCTACACACCTCTACAGCTCAGCTCCCTGAGAGGCCAGGCCTGCTATTGG GCAGGTGCCAGCCAGGCAGTGGGGGAGTGGACCAGGCTGGGATGGCAGAGAGAGGCCAGC TCAGGATAACGGACAGAGTGAACCCTGCCGGGGCATGCCGTGAAAGCTAAACCTGGGTCC TCTGTGGGGCTGCAGAGAAGCCAGAGTCCTGAGGAAAGGCCCACATTGGCCAATGGCACT GTGGGAGGAAGGTCAGGGGTCTCAAGGCAGAAAATGGATACCAGAAAATAGTTGATAATT TTCAGTGTTCTTCCACATATATTATTCCAGTTTATCCTTTCAGCAGCTTCCAAGGTTAAA AACAATAGGAATTTTCCTTTGCAGTTTATATACAATATTTACTATGCAGAAGTGCAAGAC ACTATACAGTGATCTCATTTAATCCTCAGAGCAGTGAACTGTTTATATGCAGTGGCTATG GTGGATTTCTACAGAAGACATAGAAACAAGGCTGCCCTCAGAGTTAATTAAGAAACCTAC AGGAAATGGGTTGCCTAGCCCCTTGGAAGAGCAAACCAAGAGCCTATCTTTAAATGTGGG TCTACAAAGCCAGGGAACTTGCAATCTTCAATCGAAGTGGAGATAGACTTGGAACTGATT AGAATTAAGTTGTCAGAGCTCTAATCCATGGTATGGGTTGTTCACATGGATGTCTGGGAT AAGGAGAGCCCCAGAGGTTGGAGTTCATGGAGAAATCCTGGGAGTAAAAGAATTCCATGT ATTCACAAGGAATGCAGGTAAATAAAACAGGAGAAAAAGAAAAAAATTTAAAAAAATAAT TCTGGACTTCAAACGAGGCATTGCTGTTTCATTGATCAGGCCATCTCTGCTAGGGTGGAT GAGAGCACTTTACGCTCCCTGAGCTGACAGATGCTTCTTGACATCGACTCATCCTCCTAT TATGAGAGAGAGGAAGGAGTTCAGCATTCCCACCTCAATACTCACCAGGCAGTCATTTTT AATCTGGCCTTCCTTCATAGGCCATCTTCAGGAGGAGGCTAGGCTGACTCAAGTATTGGG CTACTTGACAGTTATATTTCTCCTTTTAATAAATATATTGACACCTGCGTAGTTTTAGGC AACTGACTTCTTTCTGACCCCCAGATTCCTTATCTGTAAAGAAAGAACATTGAGTGATTA AATTATAGTGTTTGTAAAGCACTTAAAGGGAGAGAGAAGAACACAGAAAGTGCTCAGTAA GTGGAAAGTGTTATTTCCACTGTTACATATAATTAATCAGTGAACTTGGCTAATGTGAGA CCTCCCTTGATAGACAACTCTGCAGATGATGCCAACAGGCTCAGGTTTTAAGTCTGCGTT ATTGCTACTGACTCTCATTTCCCTGGTATGAACTGGCAAAGAGCTTATAGTATCGTGTCT GGGCAGTCAGCTGCCTTCCCCAGGCCACTCCCCTGTGTATGAAATTCTGACGTGAAAGTA TTAGCGTCCCCGTTTTATGGATAACAGAAACCAATACCTAAACAGACCAAGTGTCCAAGA TCATCCTGCTAATAAGAGGAAGACTAGGTCTTGAACTCTGATCTCTGACATAAAACTCTG TGGTTGTCTCGCAGCCCTGGGCTACCTTGTCTGCAATAAGCAGGGGATGACCTTCTCTGT TCTTGCTGCAGCCACTGCCCTGATGGTTATATCTGCCTTAAAACTTCTGACAACCCGGAT TTTAACTACACCAGCTTTGATTCCTTTGCTTGGGCTTTCCTCTCACTGTTCCGCCTCATG ACACAGGATTCCTGGGAACGCCTCTACCAGCAGGTACTGAGCTGCATTCCCCCACGCAGA CTTGGTTGGAGGCTGGTGGGTCAGCTGCATGGCTGTGCTTTTCCTGGAGGAGGCTGACTT AAATTCAGGGGTCCATGTGCTATGGGTACAGGTTTCTCTTCTTTCCCTTGAACCTGCAGA CCCTGAGGACTTCTGGGAAAATCTATATGATCTTTTTTGTGCTCGTAATCTTCCTGGGAT CTTTCTACCTGGTCAACTTGATCTTGGCTGTAGTCACCATGGCGTATGAGGAGCAGAACC AGGCAACCACTGATGAAATTGAAGCAAAGGAGAAGAAGTTCCAGGAGGCCCTCGAGATGC TCCGGAAGGAGCAGGAGGTTTGTGAGGAGTGGAAGCTTTGTGAAGAAGGTTGCAGACTGT CCATTCTTGGATACTGTTCTGGACTAGCCACTTCAATTAGGGCACCGTTTAGGGAGCTAT AGGAGGAATCATCTTGCCTCAGGGACCATATTAGATCAGGTTTTCTCAAAGAAGAAGCTA AGTGAGGATTCTTGTGCAAGTGACCTATTGAGGGAGTGCTCTCAGGAGAAAGCGAATGAA GCAAAACAGAATGGGGCAGAGGGTGGAACTAAACAAGGAGTGTTCTCAGCTAGAGACCAG CTTCAGCTTGATCCCATGGGGAACTCTGGGGCATGACCTGTACCACAGTTAGTCCTACCT TGAGGCAAGGGGGCCAGCTTTTTTTTTTTACCCTCCCCATACCTATCCCAGAAGCAGGCA GCCAAAAGAGACTTTTTTTGTAGAATGCTGCAAGTATGAGCCATCAGCTGCTGCCATGCA AGCAGCTCGGGATAGCTGTGCTGACCTGGTGAAAGGGATCTGGGTGGGGCACCAACAGAG TACCCCATGAATGAGTGGGGGTGACCCGAGGTGTCTGTTCCTGCCATGGCCTAATCCCAC CACCACCCTGGTCCTAACCCTTCAGCTTCTCTGTTTCAGATCTCTCACCCCTGGCACCCC ATGTCTGGGGGACACATTCATGCTAAGTCCAAGCAAATACTATTGGCTACACCTGCTGTT CTCTCTTCGCAGGTGCTAGCAGCACTAGGGATTGACACAACCTCTCTCCACTCCCACAAT GGATCACCTTTAACCTCCAAAAATGCCAGTGAGAGAAGGCATAGAATAAAGCCAAGAGTG TCAGAGGGCTCCACAGAAGACAACAAATCACCCCGCTCTGATCCTTACAACCAGCGCAGG ATGGTAAGAGGCTCAGGTTTGTGCTCTAAAGATTACCCACCATTGCAGCTGCCGTAGACA ATGAGTTGGAGTGGGAAGAGGTGTGTGGCAGAGAAAGAGCTGGAGGCATAGAGCCTAAAA GGATCCAGACACCCCTCCAAAATGGTGTCTTAAACTAGGAAAGTGTAATTATAGATGGGG AAAAGGGGTGAATTATAAGGACACCTTAGGGAAGTGGATCAACAAGATTGGGATTGTGGA CTGTGGAAAAAGGAAAGGGGAGTCCAATTTGACTCCCAGGTTTCAGGATTGGGCAATCAA ATGGGTGGTGATTCCAAGATGAGGACACTGGAGGAAGAACCCCGGTTTGTAGGAAAGGTG GGAAGCCCATACTGAACATGCCAATCTTAAGGTGTCTGCGGACATCAAAGGTCAGCTTCC TTCTGAGGCTCTGGTCTGCAGTTCTAGAGTGCAAGGTTCCCTGTCCGTGGTGCTGAGATC CTTCATGCACCTTTTTTTTTTCTGATGCGTCGAGGTGGTTTTTATTTTTTTACTTAGTTT TTTTTAATTGACACTTCACAATTTTATATAATTATTGGGTATAATTTCTGACCTCCTTCC TAAGGTCCTGCAATTCAAGCTGTCACCTGTTGTTATCAGAAAAGTTTCCAGCACCCATCT CTGTGACACAATCCAGCCTGGGAATGGCTTCAGCACCTCACCTCAGACCCTGTAGCAATT CTTTTCCCCCCATAATAACTTTCTGCCTCCCACTCTAAAATTTGCACTGCAACTTTCCTC GCCACGCTTCCTCCCACCACCTTCCTGCCCCTTGAAAGTCCTTTTACCCCAAATATGCCC TTTCTATAGGTCTGACAGATAATATCTGCTGTGTGGGAGGGTGGCTCCACACTAACAGTG TGGAAAAGGGGATCAACATGGCTATTTTTTTATGCTTTATACCTCTCACTTCCCTCTCAA TCTCTTTTCCTGTGCTAAGCCGAAGGATATTTTACTTTTTTGCAATACCATCTCTCCAAC CCTCAGATTTATGTGTGCATATACAAACACAGACACACTTGCATGCACACTGACCTCATG ACACATCCACACTTCAGCATTTATCTGCATCTGTGCCCTCCTCTTTCTTGCTTTGCCCCT TTTGAGCAGCACCTGGCATACATGGGGCACAGTTCAGACAATCACAGAACAGGATGAATC ACAGCTTCCATGGAAATGTCAATGACACTGAATTGGGCTTTAATTTCCCCCATTCTGTCA GCCCCACACAGATTAGCACTTGGATGTCCAATAATTGGTATGTGTGCTTGTCTCTCCAGT GATGCTTACTTTCAAAGGGAAAAAAGAGGAGCTGTAACTTGTTCATTCAACAATACTAGG TGCTTGGTATAAAGAGATGACTAGAACATGTTCTTTTCCCTAAGGATTTCCATTTGGGTG GAGGAGGCAGATATAAACACAGCTATGTGTTAAATAGTGTATTAGAGACACATACATGGT GTTTTGAGAATCAGGGAAGAAACATCCACTCTTATGGGGCTGGATAGGTATCAGAAAAGG CTTCCTGGAGGAAGGCATAATTGGGATCAACAATAAATAGCATGTAGGGGTTCTTCTAGT AATTGAAGACTTGAGAGAGTTTGGGGAGGAGTATTTTCCTGGCAGAAAGAACAACATGTA TGTGCAAAGACAGGGAGGCACCATTTAAGTACTTCTTTAATGCTAATTGATTTATTGGAG CCCACTCAGGAAGGCATCCTCTCCCTCTTTTCCCAGGTACATTCATGTGGTAAATAGACC TAATGCAGTTTTTATCTAAAGCCATTTTATAACCAATAGAAATTAAATAGAGGAATATAT AGAAGAGACCCAGGAGGGTTTTTGTGGAAAATCACAGACCAGTTGGTCTCGTTAGAGGAT GAATTTTAAAGCAATTCTAATGCTTATACTCAGAAGACATTGCCCACCATTTGTCAAAAC CTTAATTCTAAAATAACTCGTGGCCAAGGAGACACTGCTGTCAAATATGCAACCAAAATT TTCTTCTCAGTTTCTTCATTGGTTTGCTTGGTGCTCAAGTGCAAATAATGTAGTATTCCC TTCCTTAATATGGATGTGTCAATTTTTCTCATTTGCTTCAAAAATTGGAAGGATTTTTGC ATTTTATGAGTTCTATAGTCCTTGGGCATGTATTACAGGCAGAGAGTACTCTATTTATAA TACTGAGCTTTTAATAATGGTTGCTAAACAGGATCAGGCTTCAAATTATCAAGAGAGGAA ACTTCAGTTCTGTGAGATAAGGGGATTAGTAAGGAGAACCCCAATTGGCAAGATTCTCCA CTTTAGTAGCTTAGTAATAAAAAGGAGTTAAGGAAGGTGAGTGGAATAAATCGGTATGAT CAGAGTTTGGTATGAAGTCAATGTTTAGATTTTGTACATACTATAGACCACCTGAGGACA CAAATGATGGTTTATTATGGTCAAGTTTCACTATGGTCATGGGAACTACCTAACCCAGCA CACTAATTCTCTGAGGGCTCTGGGTTTATGAATTAGCTCTTTTCTCTGGCAACTTTAAGC CACACAGTTCCCTCAATGGAAGATTTTGATGGGAAATGTTTATTGATTTGGAGGAATGAC ATTATTCTTCCAGGAAATTAGGCTGCAACCCTTCCTTCAGTCTCTCCATTGTTGACTGTT CAAGCCTCGAGGGATCCTAGAAACTCATTGCACTGTACAGCTCTTGTCTCCTCCTTTGCA ACACAAACTCTCACATTCATACAGATGGGATCTAATGAATTGTACTACCTAATTTTCAGA CAATGAAGTGCACTTATTCATTCAACAACCACTTACCAAGTATGTTCCAGTACTGGTGTG CATAGTAGCCTCCTTAGGTCACAACTTTGCATGTGGAAAAGTGAACTTAGGACAGCTTTG TCTAGCCCTGTGGCCAAAGGTTGGCAATCAACTGAACTATCCAGGTGACAAGGTTTCCTC TGACATGTTCCTGACCATGATAACCTATTTGCTGAGCATCAGCACGCTGTAATCTCTCCT CTCTCCTGGTCTTTCCCGGAGTCTATCTAGAGTCTCCTTTTTTCTTTTTCCCCACCATGC ATACCTAGAATACCCCTTAGATTTGTGCCTTTACTTAGTGACTGAAAGGTCATAAAGACA GGGGCAGGGAAATGGGAGTAGGTAGAGGGAAGTGTTGGGCTCTAGATTTCCCAGAGGACC AGTTTCCAGCCTTCTTGCTCCTTTGGGGCAATGCCCAGTCTTTTCTAGGCCTCGCCTCTG GAAAACGCCGGGCTAGTCATGGCAGTGTGTTCCATTTCCGGTCCCCTGGCCGAGATATCT CACTCCCTGAGGGAGTCACAGATGATGGAGTCTTTCCTGGAGACCACGAAAGCCATCGGG GCTCTCTGCTGCTGGGTGGGGGTGCTGGCCAGCAAGGCCCCCTCCCTAGAAGCCCTCTTC CTCAACCCAGCAACCCTGACTCCAGGCATGGAGAAGATGAACACCAACCGCCGCCCACTA GTGAGCTTGCCCCTGGAGCTGTCGATGTCTCGGTGAGTTTGTGAATGCTTCAGACTCCCT CAGGCTGTCTTCCACCTAGAAGCCTTGAGCCAAGAAGCCTGAGTATCACCAATGGAAGGG ATCTTTAGCCATCATCCTGTCCAATGCCTCCCCATTCTACTCCTTTTTTACAGACATAGA AGATGAGACCCAGAGAGAGAGAGAGATTGAGTTGGCCTGAGGTCAAACAGGTTTCCTGGT TTATTCTGCAGTAGTGATGCAAATTATATTCCATTTGACCCCATATTTCTTATCAGTGCT TCTCTGTCCCCACTCAACATCACTGCCTCAGTGATAGGCCTGCTTTCTTGTTGGACAGGT ATGAGAAAAACTGAAGGACCCTTCTCTTCCTGCTTATGGCTCTATGCAAACTCTTCTAGG CTGGGCAAATGGCACCCAGAGAAATTCAGGAAGTTTCTTAGGACAGATCTGAATAGAAGC ACAGTATTCCTTGCTTTACTCACAGAAGAAATAATTTGCCAAAATGAGCAAATTAGGCAA CAGTGACTAACTGGCCAGCACTCTTTAATAGAATAATCGCAGAGTGTAATGGCAAGTCAG GGATTCCTCATTGGATTGGGAGCTGTGAGTTAATGTGAGCTCTCCCCTCCAGTTTCAGCA ATGATGTTTCTAGTGTTTTTCATTTGCCCTCTCTGGCTAGCCAGGGATACAGCGCAATTG CAGACAGAATTGTAAAGAGACCAATAAATGGTTCCACTGATACAGATAAGTCTTAGAGTT GCCTTAGCTCAGTCTCTGACTCAATGTCATGTGATTCAGGGTCCCAGCAAGAAACAGATG GACCACTCCGATGGGCTAATAGAGAGAGTTTAGTAAAGTATTTACAGAGGTGTGGGCTGG GCTTAGGGAATGAATAAGGGACAGTGAGGCACCTAGAAACTAGTAACAGGGGGAAGCTGT TACATGCCTAGGCTGAAGGGATGAAGGAAAGGACCAGAATTAGACTACAGCAGCAGCTGC CCTCATGTGTCAGAGAGGCTGCCTGGCAGAACTTGTGATCTTAGTAGAGGATCACAGCTC TGCTACTCAGCCATAGCACAGGAGAGAAGAAGCCAGGGGAAAATACCTGACTTGTCTCTC CTTCTATCCTTGAATGTCCTGCCTATGTCTCCCATTAACCAAACCTGGCCAGAACCAGAG GGTAAGGATAACACATTTTGTACAGGTCTCCCTCCCCAGGAACAGAGACAGTGGAGAAGG TAGAGAGTAGGTCTCCAGGGCAAATGGAATCACCAAAACAGAGCCTTAAAGTCAAAGGCA TCCTGCAGGGCTCGCTGGGAGTGCATTAGCACTCTAGAAAATGCCTCACTGAAGGCATTT ATGCCTCTCTTGTGATGGGCCCCCACCCTCCTTTCTCTGTTCCTGAGATTATCATGTGTC TAGATATTCTGACCTTGATTCATTCTAGTTAAATTACCAGGCTAGTGAAAGATTGACTCT TTAGGCCCTTCTGTTTAGTATGGACCTATATTTTAGTCCCAACTGTGCCAGTTACCACCT GAGTCACATTTAGCAAGTCCTTTAAATTATCCAAGCCTCCTTATCTCCCCTTGTAAAAAG GTTTCTAAATAGCTGGAGGGTTAAATGAAATAACAAAGTGCCTACATTTTTATACATGTC TGACAGATAACAATAAATAGTGACTACTAGTATCATTTGCTTAGTCCTCATGGTTGGAAG CTTTATAAACTTTCTTTATGTTTATTAACTGTTTATACTTACAGTATTCAGCAATAACAT GCTGTACAGATTTGTAGCCTAGGAGCAATAGGCCACACTATTTAGCCTAGATGTATAGAA GGCTATAGCATTTGTTTGTGTAAATACAATGTAGGATATTCACAGAATGATGAAATCACC TAATGATGTATTTGTCAGAACATATCTCTATCTGTAAGTGACATATGACTATATATGAGA TTGGCCAATAAGAGGCTGAATGATCCAACTTTGCCATGAAGATGTCAGGTTAAAGGAGCG TCCTGACTCTGTTTGTGTCTCAACAGGCATTCGATGCAGGACAAAAGAAGACTTTCTTGT CAGCAGAATACTTAGATGAACCTTTCCGGGCCCAAAGGGCAATGAGTGTTGTCAGTATCA TAACCTCCGTCCTTGAGGGTAAGTGCTGCTCACTGCATTGTTCATCCACTGTGTCATGAT GAATTCCCATGTGAGCAGCAGAAGCAATGTCTCTAACTCGTGTCAAAGCGTCCATCTCTG TGTACTGGAATTCCTAATACTCCTCATTCTTATGTCTTAGGCCCCACCATCTTTTATCAA TGTGGTTTTTTAGATGCATAATTTTTCAAAAACAAAATAATTTGATGAATAAAATATCCT AGCTCATCTCTTGTTCAAATTAGCATCTGCTTTGCATGCATCACAAAACTAGATTTTTAA AAGTGATCTGGCAGGCATCAGAGAATTTCAGGCCAGCTCTCCGGTGTACAAGAAATTTAA CCATCCTTAATTTTCCAACATGGCAAAATTATCCTTTTCATGGTTTCATAATTATATTTT TCATGGTTGTAACTTACACAGTTGTAATTTAATTTCTTGTTCAAGCCCACATTATGCTGA ATATTGGTAGACAATTTAAGTCCATTAATCAGTCAATTTAACTAAGTCCTTGTTAATTTA GAATTCTACCAATCAAGTGGGAATCCACTCAGTCTCCCCAGCAATGTATCTAAGTTCCAA GCCAAAGGGTAGAGGAAGAGAGTGGCAATGGTCATGGCCACCTATCCACACACTTTGCCA TAGAGCGTGATTGCATCCCTCTAACCTTGAGCAGGAGGTCTGTTCTAATCCAGCCTTTTT TTGCCTTAGGGTATGGAGAATGCAACCTTCATCATTTCCAGTTATCTTACTCCCCTAGAC AAATTAAACTGACCTAGGCTGGAGGAGTTGTAAGGGAGCTTGAGTGCTGGTGTGCCCCTG AGAAGGAGTCCTGTGTGTTTTCCTCCCAGCCCTAGGCTGAATCCTAATTTGAGGGGAAGA AGTGGTGAGCAATAGAAACCAAAAAAATGTTTGAGGGATTTCAAAACAGAGCCAATGTGT GCCCTACTTTCTAGGTAAAGCCAGAGGTGGCAAGCCTTAGAGAGAGATTCACATGCACAG AAACTGTAGAGTAGAACTAGCCAGTAATTTACCAGATATGCAGAGATGTATCTGTTTTGC TCACAGCTGGATCTCCATTACCTAGAAGAGCAGTGCATTAACAAAGGTGATATCTGGGGG GTGATGCTACTTTATAGTGGTGTAAACTGATAAAAAGGAAGAAAATAAGGAATAGTCAGG TATCTTGGGGTAGGATAGACTTCCGCTCAAGGAAGATACTGTGAGAGTAGAGTAAGAAAG GCAGGAGAAACAAAGACGTTTTTAAGAAAATCTGGGGAAAGGCAACCTTAGCTTAGGTCA ACCTTAGGTTAGAGAAGGCCTGAAGCAGCCTCAGAGAGAGCCCTGTGGCTCCAGAATGGC AGAGAGGAGCTGGGTGATGACTTAGCCCCCAGTGAAGGCTATGGAAAAAGATGAGTGAGA CCCATAGAGGGAAACTAGAGAGAGAAGAGCCATCTTCATGGACTCTCCCACCAGGGACCA GATGGGAGCTACAACTCAGGCAAAATACGCTTAGCAATGACAAAGGACCAGCTCAACTCT CCTCCAGGTTTGAGCCCTACAAAACTCAGACACAGACTCAGATATTGAAATAGTACCCAA CATTGACTTAACAAGAGACAGTTCCCATGGTGAGGACTTGAAGCATAAATTGAATTCAAC TAAGTTACATTTCTTGCATACCTGAGTTTGTGCTTTGAGAAATGTGTACTCATTTCTCAA CAACAACGTGTACAGACTCTCTTCTATTATTTTTTCTCATTGATAATTATTTAATATCAC TGTTCATTGTAATAGAGTCTGTACTTCTAACATTTTTCATGGCATAGAATTTGACCAAGC TGCTATTCCCACAATTTCTTTCTTGGCCAGATAGGTTTCTTGGTTTTTGTTTGTTTGTTT TGGTTTTTGTTTTGCTATTAGTCATCAAACAGCAAACATTGAATACAAACAGTTTGTTTT TTTCATTTATTCTCGAAAACTGTTAAGCTTCCTCCTTCTCTCTTCTTTCATTTATTCTCT TATCAACAAGCATTTGTTGAGCCCCTGTTGTGTGTGTGGCATTGCTTCAGGTGCTGGGAT TCAGTGGTGAACTGATATAAGCTTGCGCTCATGGAATCTACATCCTAGCAGGGAAGGGGA CATGAAAGATGAATAGATATTCAGATATAAGAAGTAGGAGAAAAAATAAAGTAGGAAAGG AGATGGAAAATTCTCTCCAAGAAGAACAGAAATTTGAATTAAATAAAGGAATCAGCTATG TAAGAAGCTGAGGAGAAAGTTCGCAGACACAAATAGCAAGTACAATGCATTCTGCAAGAA TGAGATAGCAACAAGCAAAATGCATGTCCCAAACACATCCCAGTGTAGGTAAGACAAAAG AAGGTCATGGGATGAATGAGAGGCAACAGAGGGTAGTAGTTAGAACACTGCTCTGGAACC CGGCTGCCTGGGTTTGCATCCTAGCTCTTCCACTTATGGTACACAGTGTGACCTTGGAGC TTCAGTAACTTTTCTGTGCCTCAGATTTTTAGCCTGTAAAGTAGTGAGAATATACTTATC TTGTAGAGTTTTGTGAGGATTAAATGAGTCAGGTCAAGCAAAATGCTTAGAACCACGACT GGCATGTAGTAAATACTCATCACATTTGGCTATTATGATTTACTAGGTTGTAAAATCTAT GAGGGCGGAGATGTGTCTGTTTTGCTCACAGCCTGACCTCCATTACCCAGAAGACAAGTG CATGCACAAAGGTGATATTTGGGTGGTTGGCGGCGGGAGTGATGCTAATTTATAACAGTG TGAACTGATAAAAAGGAAGAAAATAAGTAATAGGCAAATATGTTGGGGTAGGGTAGACTT CCCCTCAAGGAAGATGCTGTGACAGTAGAGTAAGAAAGGCCAGAGAAACAATGATGTTTT AAAGAAAATCTGAGGAGAGGAAACCTTAGGTTAAATAAAAGAGGTCAAAGTTTTCTGAGC ATAGGGCACATCATCTTCTCTGGTTGGATGTTATATTCATTACACACATGAGACACCTGA AGGATGCTTCATGCTGCCAGAGTCGTAATAGGATTTATAAAGGCCACCTCTTTAAGAGGG ATTATGCTTAAGAAAGTTTGGCCCAAACCAACAGGAGTTTGACAATTCACTGAACTCAAA GCATGTGAACCTTCTATGCTTGGCATGCAAGCTGAGATTTCACTGTTGTCCAAGCCAGAA TTCCAGAAGCTGGGTCACAGCAGTGAGCTTACTGGAGTGGTGAGTGCCTTAAAGAGGAAG TGCAGGATACTAAGTGAAATATGGTAAGATTTGCCTGGAGCCAGGGAATTATCCCAGGCT GAAAGGACTGCAAAGCAGAAGAGAGAATAACATTTGAAGGAATGAAAAAGCCTAATGTAG CTGTAACCTAGAGAACCAGGAGAACTGAAAATAAGGCTAGAGGGATGAGTTGGGGCCAGA TCCCAAGAAACTCCTAGACTGCATTAAGGATATGGGACTTCATGCTAAGAGGAGTGAGGT GCCATTAAAGGATTGGAGGCAGGGAAGTTACATTTGCATTTTATAAAAGTTACTCTGGAT ACTTGTAGATATAAGAGGGGAAGTATGTTGTCAGGGAAACCAGTTAGGAGGAAGCCAAGT ACAAGATGGCAGTGCTTGAACTCTGGGGTAGTGACAGTGGGGCTGATGGGAAATGGAAGG ATATGAGAGCTATCTAAGGGGCATAAGATTCAGGACTTGATGATGCATATGGTTCTGGTA TAAGTGCCTTAAGATATGCTGATACAATAACTCATAAAGGGAGCATTTTAGGGGAAGAAG GGGGCTTTAATGGGGAAAGATGAAAGAAGATTATGAGTTGAGTCTGGGATATATTGGGTT TCAGGTGCCCTTGGAGCATCTAGGTAGGGGTGTCCAGTAGGAAGTTGACTTTAGGATATA GGGGTCTAGAACTTAGATGACAAAGATGGCATATATATATATATATATATATATATATAT ATATATACACATACACACACATATATATATGTATATATATATATTTGAAAGCAGTTTGAA TGTAAGTGGTAATTTTGAAAAGATGCAACTGGATGAGTTAACACAGAAGGGTTGAAGTAA GAAGAGAAGAAGGCCTCAGACACAGTTCTAACAAACTAAATGTTTAATAGTTAAGCAGAA GAGATGGACCCAGCAGAGGAGACTGAAAAGGAAGGTACAGAAATAGGAAAAGATCTAGAA GAATACAATGCAGAGCCAAGAGTAAAAATTTCTTCAGAAAGAATAGAAAGGCCAACAGAG GCAATGCTGTGGAAAGGTCAGGTAAAATGAAACATGAAAAGGTCCAGGGGCTTTAGTCAC ATGGATGTCTTTGGTGATCTAAGCAAAAGAGGATTAATGGAGTAAAAGGGTAGGCAGAGG TCATATTGAAGGGGCTTAAAGAGCGGGTGAGAGGTGAAGAAATGGAGATATGGTGTACAG ACAATTTTTTCAAGAAGTTTGCCTGTGAGGAAAAGGAGATAGTAAGATGGCACTAGGGAG TAGGGAGAATTGAAAGAAGACTTTTTGGTTTTTGTGTTTGGGGGAGTTTTTGTTTGTTTG TTTGTTTTGAGCATATTTGCATGCTAATGGGAAGGATCTGGGAGAATAAGAGAGACTGAA GATTAAGGGAAGAAAGGGTATATTCAACAGACTCCTGCAGTGGCAGGAGAGAATGCACAG TGGAGACACTGGTGTTAACAGGAGCAGGGGGACCTCTTTAAGCGTAACAGGATGAAGGGA GGAAAGTTCTCTCTGTTATCAATTTTATATAGGAGATCCTTTTGAGGTGGGGCCATGGTC ATGTTTTAAGCTGGCAAGTAGATACTTTTAGTCTCTGTGTCTCTGCAATCAATCAAGTTC AACAATGTCAAGGCTGTTTACAAAGATGCCTCTCTTTTCCCATCATGCCTCACAGGTGGC TGCTTCCTGCCAAAATGGCACATCTAAAGTGTCCTCATTCATGACTACTTCCTGTGCTTC TTTTTAGTGCCAAAATAGGTCCAGGGAAGTTCTTGCTTTTGTTCCAAACAGGGATTGTTG ATTTTGGTCCCAGGATGTGCCAACTTCTTTAGAGAACTATGCTTTGCCAGTTTGGACATC TGTAGTTTCTAAAATCCAGTCAGTGATTAAAACAGCATGACAGTGAAAATAAATAGTCTG ATCTGTGGAAGAGAAATGGACATGTGTATATGAAAACACATGAACATAAATGGTAATAAC ATGACAAAAATGACATTTCAAATCTTGGGAAAATGAAAAATTATTCAATAAGTAATGTTG ATATAAATAATCAGAGACAGGAAAAAATAAACCACATCAATATCTTACTCTTTACCCAAA ATAAATTTCAAATGCACCAAAGTTTTAAGTGAAAAAAGTGAAACTACAAAAGAAAGAAAA AAACATGGAGAAGGTATTTTCCAAGCCTTGTAGTGAGAAAAGCTTCATATGTAAGGCATG TATGCATATGTGGCTAACCTGCATAATGTGCACATTTACCCTAAAACTTAAAGTATAATT TAAAAAAAGAAAAAAATTTAAAATAAAAAAATGAAGAAAATTGAAAAAAAAAGAAAACAA ATAAAAATACTACTGATAAATCTTGACTACATACACATTTTTAAATTATCAGGAAAAAAC ACTGTTAGCAAAATTTGGGGGCACACAGTTATTTCTCTAAACTGATTTTTCAAGTGCATA TAGTAAAAAAAACCCTAAACACTGCAAGTGTGTATGCAATGACAACTATGTCTCCTTCCC ACTATAGACCCTTCCTGCTCCCCAGACTCCTGGTGCACCCCCCTCCCACTCCAGTTTATC TTATATTCTTCAAGCAATGTTCTATGCATATATAAGCACATGCATGCACATACACACACT ACACATACATATTTTTCTACATCACCACAAATAAATCTGCCTCATTCTTTTTCACAATGT ATTATGCTTTATCAGTTGTTGTTTTCCATTATTATTATAAACAATGGACATCATCTTTCT TTGCCCACATAGGCTGATATCTGTTCAAGATGCATGTCTTGCCGAGGAATTTATAGATAA ACCTAAAGCTAAGAAATTGCCCTCCATCGAGGTGAACACAGTTACACATCCACATGGAGA TTTAACGGAGGCAGGTAGAGAGATAAGCCTAGTGTTCAGGAAGGGATCAGAAATTGGACA CATAATTTGGTGGGAATTGGCATAGAGATGGTTTTTTAGGGCTATGAGATTCAAAAACAC AGAGAAGCTAAGGTGAGAATGCTTGAGTTATTCAACATTTACAAGCAGGAAAGGAAAAGG GAGACAAAAACAGAAAGTGAAAAGAAGTGGAGAAATATGGGAGAAAAAGCAAGAGATGAT GCAGTCCCCAGAGCCAAATGAACACTGGTTTCAAAGAGAACAATATAAATTCTGTCAAAT GTTATAAGGGTAAGATGAGAACTGGAAATCAATTATTTGAATTTGGAAGCAGTTAAAGTC AGATTCCAGGGGCTGCAGAGAAAAAAAGAGGTAAGGAGCTGTAGAAGGTTAAGTGTAGAA AACTCTTGCAAGGAATTTTACTGTCAAAGAAGCAGTAATGAAAGAGAGCATGGGGTCCAG AGAGACTTTTTCAAAGCCGCGAGATAATTAAAGCATGTTTACCTGCTCATGGCAATGGTC TGGAAGGGAGACAGGTCTGGATGATGCAGGCCTTGAATAGGTGGGAGAGGAAAGAGCCCG GCACAAGTGGGAGGGGTAGAGCCACCCACGCACTGTGTGGGGCACGAAGACAGAATACCA GGTGGACTTGCAGATATGCCAGAGATTTAGTGGTAGGAAGAAGGGGAAGTTCTTTTGTGA ACTAGTCTATATTCTGAGTGAAATAAAAGGGAGCTCATCAACAGAGAGTAAGTAGAAAGA AGAGCCGGAGGTTAGACGGGGGAAGAGGAAGTGAGAAACAGTCATCTGGAAGAATGAAGG AATGGAATATAATATAGAAACACAGCCAGATTTCTGGGCAGGGCAAGGCTGGGGCTCACA GATCTCTAGGAGGGTTGGTAAGCATAGTTGTGTATTTTTTCCAGCCAGGTCCAGCTGGTT GGGTGCAAGTACAGAGTAGTCTACAAATTGGGTCTATTCACAGTTCAGGTTTTGCCAGGC AAGTACAATGAAGGAGGAGAGGGGCAAAGGAATTGAGGGTGCCTACAAGGGAGTAGTTAG AGAGATGGATGTGAAATCTAAGCTGGGCAAATTGAGAAGTAAGGACATGATATAGGTGAT GGGCAGTAAAAATATGTAATGTCAGCAGTTTAAAGGACTGGATGGGGCAGATATTAATTG GAGTTGCAGGACTAAAGGAGTTCAAAATATAGGAAATGAATACCAGAGACAGAGAGAGGG CTGAAGTCAAAATGTTGGAGGTGGTACTTATTATTAACAACAAGGTCTAGAGGATGACCG CAGAATTGGGGTCCAAGGTGACACATGGCTGACAGCTGTCATTGACCACACTATAATGCA GAACTCGAGGAGTCTGAACAGAAGTGCCCACCCTGCTTGACCAGCTTGTCTCAGAAGTAT CTGATCTGGGATTGCTGCCCCATGTGGGTGAAGCTCAAGACAATTCTCTTTGGGCTTGTG ACGGATCCCTTTGCAGAGCTCACCATCACCTTGTGCATCGTGGTGAACACCATCTTCATG GCCATGGAGCACCATGGCATGAGCCCTACCTTCGAAGCCATGCTCCAGATAGGCAACATC GTGAGTGCTCTGGCTGGTACTGCCCTCACTGGCGTGGTCGGGGCGGGGTGGGGATGATGG CAGGGTGCAGTTCGGGTGGGGTGGGGCATGCCCTTATTGGTTAGTGTGTTGAGCCAGTTA GCTGAGAGCAGAGCAGCAGTGCAGGAGACATGTGGACACTGGGTTCAGGAGGTCAAGTGT GGGAGGACCCAGGCTGAGCTGGTGGGAATGGCTTTGGGATGAACAAGCACAGGAAAGTGA ACCAGGAGAGCCTGGAGCCAGAGGCAGCTAGTTGCCCTTTACAAAACATAGGACAACCAC TTGAATTTTCTGGATCTCAGTTGCCTCTACCTTGAAATGAGGATAAATGTCACTTTTTTA TCTAAAGACAATCTTTGGACTTGTCTGTTTCCTTCCAATGTAATTACTTTTATGACCACA ATTAACAGTAGCCACGATTTTCAATGTTGTCACTTAAGGAGAAAAAGAGTTGGGGAGGGA AGGGGAAGACAGTGGAAGGGAGGAAACATGGAAACAAGGTTGCTGTTGTAACAAAAGCAT CAGGAAGTCGGAACTTTTCTTTGGCATCTAGTTTAGACATCATGCTGCTTTAAATTTTCC TCCATCACTTGTTAGTATTATTTACTCTTAACAAGATACAATCCATACTGGATTTCACTA TATACTGGATAAAGTATCTGACTCAGAGGGACAACAACTGCTCCAACCTTGCCCCACCCA CAGGAATAGTAATAATACTCAGATGCTTCTCATGGATTATCTCATTTTATTTTCACAACA ACCCTACATGGTAGGCACTCTTGTTATTCCCTTTCCCAGATGGAAAAATGGAGATGCAAA AGGTTAAATAATCTTCCCAAGATCATAGAGTTGAGTTTAGTGGCCACATCAGAATTTGAA CCCAGACAGACCGGCTGCCAAGCCCGCTTTTCTTAACCCCTGAATTAAGCCACCTCACAA GACTTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTCAAACACACGCACACATG ATAGGCAGAAGCTAGAAAGGGTCAGCATCCTGCCTTCATGTCCGCAGCAGGGTCCACCAA GCTGGAGGTGACCTACTGGATTCACCAGAACTGTCTGGCTTTGGGGTCCAGCTTCTTATA GGCTGCAGACTTAGGTGGAGGGGGAAGGCCCAGAGGAAATGTGACTACTTGTCCCTAGAA ACCACTCCCTCCGCCATAATCCAGGGCTGTGAAAGGGGCAGCAGTAGGTTCTGGAACATG GTGGAATCAGCATGCCTCATGTTTTGGCAGCACCTAGGCTGAGGTCACATCCTAAAGTTG ATATGAAAAGGAAAAATGGAAAGAGAAGAGAATGCTTATTCCCAAGAAGTCAGCCTAAGA CCACAGAGGGCCTTATGGAATAGACATTTGAGCCCACACCCTCATTCTACAGTTAGAAAA GTGGAAGCAAGAGAGAGAAAAGGCCTTTCCCACAGTGTCAGAGGAAAGTGCCACTTTTTT AAAGCAAGAAAGTCATGTGCTTGGTGCCTGCAATGAGTCAGGTGCTTTATGAATGTTATT TCAGTGTCACAACAACATTATGAGGTTAACATCACCCTCACATGACAAAAGAGAGCACTA AGACTCAGAGAGGTTAAATATCCTGCCCAAAATTGTATAGCTGGTGAGGGTGGATCCAGG GTGGACCCAGGTCTCTGGAAGACACAAGACCTCAACCCTACCCCCCACCACACCACACTG ACTTGCCTACAAGGGACACTCCATAGATACTCTTTGAATCAGTCTTGTGGCATGAGAAAG TCACTTAAACTCTCAGCCTGTTTTCTTCTCCATAAGAAGGATGCATTGTCTGTTTCTCAG GCTGGTGAGGGGCTCAGATAAAAAGATATGAAGGAAAAGCACCTGACCCTGGTCGTTGCT TTCCTCCTTCTCTGTCTCCCTTCCCTGCTGGTTGTTGTGTTACCCGAGCCTGGTTCTGAC CTCTCTCAGTGTCAGAATTCTGTCCTATTTAAGACAATTAGAGACACACAGAGCCTAAAA GAATCTTAGAGGCAATGAATCATTGTTCTTACTAGTCACTGTTACTTATGAATGAATTAC TGTCTGCCAGGCCCTTTGTTAAGAACTGCACATGCATTGGCTCATTTTATGCCCATTATA CAGATGAGGAAATTGAGGTACAGGAAGGATGCATTATTTGTCCAAGCTTACATAAGCTAG GAAATGTCTGGGCTGGGATTTTAATCCAGGTCTGTGATGTCCCACAACCCACACGTTTTA ACTACTGAACCAGACTGACTCTTCACTGACAAAATGATATTATAATAATGATCTTAAAGA CTATCCGCTTATTCCACTCCCACCACCATTCACGTGGAAAAACTGCAGCATGCACAGCCC CTGAGAATGGTGCAGAAATGTCTCCATTCTGTCACGTTGACTGTTACCCTGGCCAATCCT CAGGAGCCCACAGATAACATAGCAACTGAACAAAACAAAAGGATTTTCTATTTTGTAAAT AAGACATTTTCTTGCTGCCATTTTTATCATTGCCTCCGACCCCACAGATCCCACTGTGAT GAAAGCTTGTTTCCACTCCTAGGTCTTTACCATATTTTTTACTGCTGAAATGGTCTTCAA AATCATTGCCTTCGACCCATACTATTATTTCCAGAAGAAGTGGAATATCTTTGACTGCAT CATCGTCACTGTGAGTCTGCTAGAGCTGGGCGTGGCCAAGAAGGGAAGCCTGTCTGTGCT GCGGAGCTTCCGCTTGGTAATGTTTTTCTCTTGATGGCTTGGGGAAACTCCCACCCAGAT TCAGGGAAGACACTGTGCTCTGGGGACTGCACCAGCCAGCTCCCCCTTCCTCTCCTGTCC TTGTGGGAGGGAGCAGGGTCTGACTCCTGGGCGACTGGAGACTTGGGTCTGTGGCCCCAG CTTTTCGCTGACTCAGAGTGGGGACATGCCTTCTCCTTTCTGGGCCTTGGCTCCCTTATC TGCACCAAGAGGGGACTTCCAAGCACTGTCCTCGTCTCTTATCAAATAACAATAACAGCA ATAATCACAGCAGCTAATACTTACTGAGCACTTACAATGTGTCAGGCATAGCACTGGACA CTTGAAAAAACGCACCTCATTTTATGCTCACAACAACCCTGAGAGATAGTCGTTTTTAAG ATCCTCAGGGGTGGGTGAGGAAAAAGAATGGTTCCATGATGACCCGAAAGTCTCCCATCT GCCAAGCAAGCGAATAAGCCACAGACCTGGATCTCAAATCCAGGTTGTCTGACTCCAGAG CCCCAGCTCCTTCCAGCCTCAAGGAAGTCATTCTCTGATTCCCTTGTGCCCGAAATCACA GCCCCAATGTCTCAAGCACCTTCAACCTGCCTATTAGTCTCACTCACATGCAATGATGGC TCTTCTCTAAACTCAGCTCCAGGACGCCCTCTACAGGAGAAGTGCCACAGGCCACCCACT TTTATCAGAGCCACAGGTTTGTCAGCCAAACCTGTCAGATTCCCAGGAGTGTCGGGTCAC TAGGTCTCCAGGCCATCTGGAGGAAGCTTGGCATCTCACTGACTCTTGGGAATAGGAAAG AGGCCTGAAGAAATGTCACGGCTTGTTAGCGCAGGCCGCCTGCTCTCAGGAATCTCTCAG TATTAACTGGGAGCCCTGCATCTAGACTTGAAGAGAGGTGACAAAACAAAGGGGAGTATC CAACAATCTTCCCAAGGTCCTGACAAGTGTTGGCATCCAGAGGTTGAAGGGGGTGCATGG TTCAGGAGGCAACGAGACTCTAGGACAGATCTGCTTGGGACCCTATCACAGGGGCCAGTG AGACAGTGCTGAGCCCCTGAACTTGGCGTACCTGGATTAGGGGTGGCAAACTCAAATGCC CATGTGCAGTGACATAGAGCCAGCCATGGAAGAGATGTGGGCACTGGATATTTGCCTCAG GTAGACAGGCAAAGATGACTCCATTTCACCTAGGCAGACCCAAAGCAGTGCACAACTCCT GGGAGGGATGTTTACATAGAAATCAATGTGACCATCTCAGGCCAGGTGCAGTGGCTCACA CCTGTAATCCTAGTACTTTGGGAGGCCGAGGCAGGCAGATCGCTTGAGCCCAGGAGTTCA AGGGCAACATGGTGAAACCCCGTCTCTACAAAAAATGAAAAAATTAGCCAGGCATGGTGG TGCACATCTGTTATCCTAGCTACTCAGAAGACTGAGGTGGGAGGATCACCTGAGACCAGG GAGGTGGAGGCTGCAGTGCACCATGATCGTGCCACTGCACTCCAGCCTGGGAGACAGAGT AAGAACCTGTCTCAAAAAAGAAAGGAGACACAGAGAGAGAGGAATAAAGATAGAAAAAGC GAGAGAAAGACAGATGAAAGAAAGAGAGAGAGCAAGAGGGAGAGAGAAAGAAAAAGAGAA AGAAAGAAAGAAGAAAGAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAGGAAGGGA GGGAGGGAGGGAGAGAGAGAGGGAGGGAGGGAGGGAGAGAGAGAGGGAGGGAGGGAGGGA AGGACGAGCTATGGCCTGTCCATTATTGTCAGAGTTACTAATTTTCCAAAAAAGTCAGAA ATCCAGATTTTTGTGCAAATGTCTTTAATATTGGCTACTAATCCAGTTTTTAAAAACACT CTAAAGACCAAATAAAGCATGTCCTGAGACACAGCTGTGCCATGAAACGCTGGTTGATGG CATCTGCCTAAGCTGATGGTACTGTCACCATCCCAGGGAGGAGAAGGGTAGAGTAGGCCT GGTAGGACTCTTTATCCATAACTTGAAAATCTTACAAGGTCAGAATTAAATCACATTTTT AGAAGGACAGATTTCTCTAGGTATCTAGAAATTGATGTCAAGATGTAGGCCCCTAAGCTA ATGGTCAATATAAGGTACCTATGCACAATCGTCAAGAGAGCAGGAGATATCACACCCCCA TACCCTGGCAGGCCCACAGTTAGTTTTCTAATAAAATACGAGGCTGGGCGCGGTGGCTCA CGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGATCACAAGGTCAGGAGATCG AGACCATCTTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCG GGCACGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCG TGAACCTGGGAGGCGGAGCTTACAGTGAGCCGAGATTGCGCCACTGCAATCCGGCCTGGG CTAAACAGCGGGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAACGAACATTTCTGC TACACTTGGGGAAGCAGAAGCTTAGTGTTTTAAGGTCTATGTGCTACTTTTGTTCTGATC CCACAGTCTAAAGTCCCAAACAACCAAAAATAGGCAAGTTTACTTCACAGTCTGCCCCTA CAGGATTCTGCAGGGGAATGCTACCCTGTCCCCACCTCGCTGATTTAACATGCTGATCAG TGCTCAATTCCCTAACCACACCCACCAACTACACACTTTCTTGGGATCTATTCCACTTCC CTCATGACCTAGCTTAGATTTCACAGCCCACCAATATTATTATGCCCTTGAAAACAAACT CAATCACCTTTTCCCTCTGCCTCTTTGCGTTTTTGTGTTTGCCAAAACCTCACCGGAGAG TCTAAGTCAAATATTTCTCTCCACCTTGTCTGCACCTGAGCTGTGAGAAATGCTGGAGAA AAATCACCGCCAGCTGGTCAGTTTCCTGGTTTTAATTAATAGACTTTATTTTCAGGACAG TTTTCAGTTTTCAGAAAACTGGAGCAGATTGAACAGAAAGCTTTCATATACCATCAATCC CACAACACACACACACACACACACACACACACACACACACACACACACAGAGTTTCCCTA TTACCAACATCTTATGTTAGTACCTACATTTGTTACAAATAACTTCACTGGTTTCCCATT GCTCTGAGAATAAAATCCAGACTCCTCATGATGGTGTACAAAGTTCTGCATAATCAGGAC CTGCCAGTTCACCAGTCTCCGTCTTGCCCACTTTGCTCCAGCCAAAAAGGCCTACTCTCT CTTCTTCACACATAACAGAGAACTTCTTGGCTTTGGGCCTTTGAAGTTTTTTTCGCTGTG CCTGGAATACTTTTCCTTTAGTCTTCACTTAGCTCACTTATTCTCAAACTCTGTGTTCAA TGCACTTGTCACCTCCTTAGAGGGGACCTCCCCAACCACACTATCTAAAGTTGGATTCCT TCATCAAATTAACCTGGCATTTACAATTGTCCTGCTATCCCATGAGGCAAAAACCAGAGT TTCTTGTCTACCGTTTTATCCCAAGGGCCTAGCACAAGGCCCAGCACATACATAGGAACT CAATAAACACCTGTTGATTGATTGAAGGAAGGAAAGAAAGAGAGGAAGGTGGAGGGAAAA AGGAAGAAATAAAAAGTCTGTCTTCCTTCTCTCCCAAAATAGCATTGCTTCTCCTCCCGT CACTTAAAGCCTTGCTCCATTTGCTCTGTTAGTCCATTGGTCTATCCTGGCACCAATATC AGTCTTAATTATGGCAGCTTTAAATATATCTGTGTCTGTAAATCAAGTAATCCACCTAAT TTACTTCAAGAATATCTTGGCCTGCACACTTTCATATTCACTTTAGAATTATCTTGCCAA GTTACATACCCACATACACACAAAACTGCTATTGTTTTTCCCATTAGGGACTGCTGTAAT TGATAGGTCAATTTGGGAAGACTTATAATCTTTACATAATTCAGTCTTCTGATCTATGAA GATAGTATACGCTGTATACCAGTCTTTCAATAAAGTTTAAAAATGTTTCCATGAGGTTTT TATAATTTTCTCCATAGGATCTTGCATATCTTCTTAAATGTTCTTTTCTGGCATTTTTGG CTAGCATTTTGATATTTAACATTTTTGTATCTAGAAATGTCTCTTATTAATTCTAATAAT TTATCTATACAATGTTTTAAATTTTCTAACATAATCACATCATGTGAAAATAACAATGAG TTTAATTTCTTCTTTTCTTTCTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTCGCTCTG TCACCCAGGCTAGAGTGCAGTGGCGTGATCTCGGCTCATTGCCAGCTCCACCTCCCGGGT TCACGCCATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGGCACCCACCACCAT GCCTGGCTAATTCTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGG TCTCGATCTCCTGACCTCATGATCTGCCTGCCTTGGCCTCCTAAAGTGCTGGGATTACAG GCATGAGCCACCCACGCCCAGCCTAATTTCTTCTTTTCTAATCCTTATGCCATTAGGTTC TTTTTACTAGATCACTGTGCTAGTTAGGACATCCAGTACACTGTTGAAAAGAAGTGTTAA TAACAGGTATCTTTATCAATTTTCTGATCCCAAAAAGAATCCTTTCAATATTTCACCACC TCTAACAATTGTGGCAGGCTTTTTTTAAAGATAAGAAATCTTTATCAGTTCAGGAAGTTC TCTTTTATCAAGAATATTTATCATGAATGGATGTTTAATTTTATCAGACAATTATTCTAC ATGTATTGAGATAATCATGTATTTTCTCCTTCAATCTGTTAAAGTGGTGAATTATATTAA TTGATTTTCTAATGTTAAATCAACTTTACATTCCCAGCTCAAACCTAGCTTGATCGTGAT GCATTTTTTAATACATTGGAAGATTTAGTTTGCTAAAAATTTGCTTAAGATTTTTTTATC CATATCAATGAGTAATATTTGATCTATAATTTTCCTGACTTTCATTGTCCTTGCCAGGTT TTGGTATCAAGGTTCCAAGATTATGCCATTTTTTTTTTAAAGCCAAGGGGGCCAGGTGGG GTGGCTCACGCCTGTAATCCCAGAATTTTGGGAGGCCAAGGTGGGAGGATTGCTTGAGGA CAGGAGTTGGAGACCAGCCTGGCCAACAGGAGAAAACCCCATCTCTACTAAAAATATATA AATTAGCTGGGTGTGGTGGCACACACCTGTAATCCCAGCTACTCAGGAGGGTGAGGCATG AGAATTGCTTGAATCCCAGAGACAGAGGTTGCAGTGAGCTGAGATCATGCCACTGCACTC CAGCCTGGGCAACAGAGCGAGACTCCGTCTCATTGAAAACAATAAAAAGAAGGCCAGGGG CAGTGGTTAATGCCTGTAATCCCAGCACTGTTGGAGGCGCAGGCAGGCGGATCACCTGAG GTCGGGAGTTCGAGACCAGCCTGACCAACATGGAGAAGACCCATCTCTACCAAAAATACA AAATTAGCCAGGTGTGATGGTGCATACTTTTAATCCCAGCTACTTGGGAGGCTGAGGGAG GAGAATTGCTTGAACCCGGGAGGCGGAGGTTGCGGTGAGCTGAGATTGCACCATTGCACT CCAGCCTGGGCAACAAGGGCAAAACTCCATCTCAAAAATAAATAAATAAATAAATAAATT TTAAATTTAAAAAAGAAAAGGAAAGAAATTAGCTGGGCATGATGGCACGTGTCTGTAGTC TCAGCTATTTGGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGGTCGTGGCTGCAG AAATTACAGCATAAATTAACTCTCCATTAGTTAACTTAATCAAAGCCAAAAGTTAGCTAA AATCTTTAACCTTCCTCCAAGACAACTTTTCATTCCAGATATCCTTTCCCAATTTATTGT AATGATAAGTATTTTAATTTTATCTTGTTTATAAAAATTTTTATTAATCAATGAAAAATA AAGCTACCTATTATACAGACAATCTCTAAAACTGAGTAACTGACCCCAATAAAGGTTTAT TTCTCACTCGTATCATAGCCTAATGCAAGCCAGACAGCTCTAGGTGGGACTCCAAGAAGT GACTTTGCCATGCCTGAAGGAGGCCAATATAACTTCAAAGTTTGCCTCCTGCTTGTGGGA AAATGGAAAGCCAAGGTTTGTTTTAAGGTTTAAACAGTGAAAGGCTTTTTCAGTCAGGGA TTTTGATTTATTTCACAATACAATTACTTCAAAAACTTAGATGGCTTCAGACCTCTCATT GCCACACAAAGTGCTTTCTTTGACAAAGTTCTCAATTGTTCTTTATTTCTTTGCTTTCCT TTCCCCATGCCTTTCTCTTGCACAAGAAACAGTGGCTACACCATTACTACAAGCTTTGCT AAGAGTTGTTTCATTTAATTGGAAAATTTAACTAGTCTTCAACATTTGAAGACTTAATCC CATTGCTCACTGCCTGGAATCTAGAAGCAGTACAGTTCCCTAAATCGTCAAGGCTCTAAA CTTCTGGATTATATTTTCTTTTATTCTTATAAACCAGCTAAATCTTTCCTGAGCTAATCT CTTTCTTGTACTGTCTTGACCAACATAGCCAATTACAACTGACCCACAATACTAAGGTTT AATAAACCCTTCCCCTAGGGTTGCTGGTTCATGAGGCTCGTGCTCTGCCTCAAAATTTTT GCCAGCAATATTTTTGTCACTGAATAACATAGATTGACATTCCAAGCCTTCTCCTATCAG CTTCCTTGTTGCCCACCACATGATCATTAAGCCAGTGCCACATAGTTGATGCTTTTGAAA AGGCAATACCTTTTTTCAATGAAGCAATTTCTCCATTACTCAAGATAAGTACAACTAGCT GCAGTGATACCCTCTAAATCTGTGAGTTAACATAATAGAGATGTATGTCTCACAGCACAT TCAGTGCTTGTTGGGAAGCTCTCCTGGGAGTGTCTACTTCAAGTAGCGCCTCAAGGATCT AGGATTATCCCATATCGTGATGTCACCATCTTCAGTTAGTGACCTTTATGGTGAACATGA AAGGGAAAGAAAAAGTTCAGGGGGTTTAAGGCTATGCCTAAAAGTCATGTATATTACTTC TGTCTCCATCTCCATTCCACCAGACAGAACCCACTAACATGACCCCAACTATTTGCAAGC AAAACTGGAAAACAGTCTTCCGTTGTGTCTGGGAGGAGAAAAGTGAATGGAGACTATATG ACCAGTCTCTTCCACATCTCACAAGACATTGTTGTTGTGTCCACTCAATGTTTATTTAGA TTCACTCTCATGCTTAGCACTTTCTTGCTCTTTATACCTTTGTTATAGAGCCAGTGGGGG CTCACTGCCCAATGCTCTAGAAGCCAATACTATGACTTTGGAAGCCAAGACGAGGACACC AGGCTTTTGAGAAAAGAAAAGCTTAATACTGCAAGCTGGCTGGCAAGAAGACAGGAGGTC AAGCTCAGATCTGTCTCCCTGTGCTGTCTTCAAGGCAGTATTTTTATTAGAAAATGTTTA GTGGGTGGATACCGGGATTTGCAGGTGATTGGTGGAAGGAAAGGGGAGGTTTGGAGAGTC CATGGGCCTGCACAGTTATCTCTTCATGCTTACTCATGGGTTGCATATGCAAATCTGGGG GGAGTTAGTATGCAATATGAAGTGGAAATTCAGTCCATGACGTCAGCAAGCTCATTCTGC ACAAACTCCAGTCGGCCATCCTGGTTCCAATTGATTTCAGCCTGTTTTTTCATCATCACA CAAGGGGAGGGAGTTTCAGTGTCTCAGCAAGTTGTTTCTTTACCTATCTGCTATCCTAAA AACTCAAGAATTTTTGTAAGTTACTGATTTCTTACTCTTTGGAACACAGTTTGAGTTTCA GATTTTCAGTGAGTTATTTCTTATCTGGTATCCTGTAAGCCTAAAAATTTAGTTATTGAT TTCTTCAACCCTTTGGAGCACAGTTTCACATTCTTGTATATTACACATTACATATTACTT CTGGGATCACTTTCTATCTACCTGAAGTACCTCCTTTAGAATTTCTTTTAGTAAGTGCCT TCTGGCCATAACCTCTTTGTTTATCTGAAATTTGCTCTTCAGTTTGCCCGTGTCTTTGAG AGACATTTCTTATGAATGTAAAATTCTAGTTGATACTTTATCTTCCTTCAGTGAAGATAG TATTCCAACAATTAGCCTCCATTGTTGCTAATGAGCAGTCAGCTTTCAATTTAACTGTTA TTCCTTCCTTTCTGGATGCTTTTTAAATCTTCTATCCATCTTTGGTGTTCTGCAGTTTCA CTGTAATATGTCTAGGTGTGGACTTCTTTCCATTTATAATGCTTAAGATATACTGACTTC TTGAATCTTTAGTTTTGTAATTTTCTTCAATTCTGGAAAGTTCCCAGACATGATGTGGGC AGTCATAGCCTCCACGCCATTCTATTTTTTCTCCTTCTGGACTTTGGTTAGATAGATGTT AAACTCTCTCATTCTATCCTCTATGTCTCTTACACTATTTTGTTATTTTTCATCTTATGT CTCTCTAGGTTACATTCTGAATAATTTATTCTGATATATCCTCAGTTCACTAATTCTCTC CCAAACTGTGTCAAATCTGATGCAAACCTTCTTCATAATATTTTTAATTTTAAGTATTAT ATTTTTCATTTCTAAGAGTGTATTTTTCACAACTTGTTATTGTTATCTATAGTTTTTTTA TTCTCTGCACATAGTTTCAAGTTATTATTTTATTTCTTTAACATATTATATCTAATTATA CTGTTATCTCTGACTAATAATTCTATCATCTGAAGAAATGGAATGACTATTTCCACATCT GTGGTTTCTTCAAGATCTCATTCACTCTTGATGACTCATTCCTTCTGTTTTTGTTTGTTT TTTTACTGCAACTCTTAAACAATTTTTGAGGGAATAGTTTGAAACCTGAGATAAAGGCAG GTTCCTCCAGAGGTATTTGCATTTAATTCTGCAGAGCACTTTGTAGAATTGCTCTAAATT AAATTCTCAACTTAAAATACCTAGGTAGTGTGAATTCCAGATAAAATCCTATATGAGCAC CACTTTGTTGTTTCAAATGCTCAGCGTCAATTTTCCTCCTGTAGCTGGTACCTAACACCA GCCATTCAATTTTCCTTGCATTCTCCTGGGATTGTTTGTGGTTCATCCTTACATGAAGGG TATAGCTCTTTGGGATCTAGTCTTAAAGCAGGGAGAAATTCCTGCTAGGCTTACAACCTT CGGCAGGCCATGGACTACGTTTCCTGCTCCCCATACTAGTTACTAATGCTCAGTTTTTAT TTGTGTTTATCAGCTGCCTCTGTGACCAGTCCACAGAGTTGTTGTTTTAAGCTCTGCTTC CCATATGAGGAGAGTAACTTCCAGATTGGGTGTAAGGTGAACGTGGACCCGCTCTCCCCC ATCACATAAAGAGGTGCATACAGCACTTACTGTATAACAGGATTAAACCTCCTCCCGTGG GATTTACTCACGGATATGGGTGATAAAATTGTAGCTACACTGTTGTGTTCAGCATTGCAT CCTAGCACCTAAGCCACAGCCTGGCTCTTAGTAGGTGCTCCATAAACACACCTTGACTGA CAGACTGACAGTTGAGCTCAGATTTTTTTTCCTGACAAATTAGAGAACCATACAAATATA TTTTAAAACACATACATTTTTATGCTTGATGATTTTGCTGTCTTGTGGGACTTAGCATAT TTGAGTTTACAGTGAAGACTTTTGAGACTTCATCTTTTCTCAGTAACTAAACCATTGGTT CCATGAGAGCAAGAGCCCACACACCTCTCTTCATACACCTCATAGGCTAGCACACTGCCA TGCACCTGCACGGCTGGAGTTCTGGGGGGTAGGTTCGTTGGTCAATTAAATCAACTTATG AATGCTGAGTCCATGAAATCATGGACATTTGCAGTTTCAAAGCTGGTGGGAGCAACAGAT GTTGTCCACTGCCCCAAGATGAGAAATGCCCTAAGAGATGTTTTGCCAATACCTATCGAA TGCAGTGGGTCATCCCCTACCCGAAGGAGGCTGGTCTCTCTTCAGTCATAAATAAGCTTG TTCTCACTTATTTGTGAGCCCCATCACACCCACCCCACCTCCACAGAAGAGGCCCATATT CAGGAGGGATCAGGCCTGTGGCTATAGAACAGAGTACAGATCGCAAGGCCACCACCCCAG TAGAAGACCCTGACTCAGCCACAGTCAATGAAGCACATTGATACTTATGAACCATAGAAG GCAGATCATATTTTTGAGTAATTTATAGCATAGGAAGAAAGGTCTCCAAAAGCAGTGTCC AGAAAACGCCCTGGTTTAATGGGATGAGAGGCCTTGTGTTTTTCCCAGATCTTTGGTTTC CAGGCAAAACTTTAGATGAAAGGTAATGAGCACAGCTACCAAAACCTGACGGTCTCTGTT TCTCTGCAGCTGCGCGTATTCAAGCTGGCCAAATCCTGGCCCACCTTAAACACACTCATC AAGATCATCGGAAACTCAGTGGGGGCACTGGGGAACCTCACCATCATCCTGGCCATCATT GTCTTTGTCTTTGCTCTGGTTGGCAAGCAGCTCCTAGGGGAAAACTACCGTAACAACCGA AAAAATATCTCCGCGCCCCATGAAGACTGGCCCCGCTGGCACATGCACGACTTCTTCCAC TCTTTCCTCATTGTCTTCCGTATCCTCTGTGGAGAGTGGATTGAGAACATGTGGGCCTGC ATGGAAGTTGGCCAAAAATCCATATGCCTCATCCTTTTCTTGACGGTGATGGTGCTAGGG AACCTGGTGGTGAGTGGTGAACCCTGCTGGGGGCACTGTCTCCTGGGGAAAGGAGAAGGC TTCTGGGTTATCTGAAAAAGCTTTGATCTGCATTTTTATGCCCAGATTGTACTTCTCTGG TTGAAAAGTCTTCAAACTTTTCAAATTCATGTGGGATTCATTACAAGAAGTGTGCCTTGG GAGGCAGTCTGACTTGCAGGAATGAGAACATATGATTAGAAAAGAAAGACCTGGTGTAGA ATCTCTGCTCCACCATTTACTAGGTGGGTGACCCCAGGCAAGAGACTTAATCTCCTCAAG ATCTAATTTCCTTAAAATCAAGAAAATAATGCTTACTCATGGGTTTACTGGGAGAATTAA ATGACAGGAGATGACATATGCAAATTGCCTAATGCAATGTCTGGGCTGAGGCAGGAACTC AATAAACGGTGTCTATTAGCTGTTAAAGGAAAAATGTCCTTCCCCAATCTGAGGGGGCTC AGAAAACATTTTTATTCAGGGAAGTAAGAGCTGACTGATTTCTCCAAGCTGCACAGGTCC TAGCTGGGAGCAGGGGGAGAGGCTGCAGGGAGGGGGCACAATGGGTCACGGACAACATCC AAGGCCACAGAAATAATAACAAAATCTACTCCATGTTTGGAGGAAGCCCCTCTGGAGACT CACTTGCCAATAAGCCCCAATACCCCTTCTCCGCCCCAGCCTGGCCCCCTGCCCTCAGCT TGTCTCCACCCACTCTGGTCATGGTGGGGTCCTTGCCCTCCTGGGACTGACTGGTTGTGT GGGAGGATGCAGTCTTCCAGAAGCCCCTTTCTTATTGCCCTGGACATCAACCAACTTGGG TTCTTTCCTCATGAGTTTTATAGACAGAAGAAAGCTAAACCAAGAAAAGGGAAGCAACAA CAAATCCAGACTCTTTAGTCTTTTCCACCCTGGTGCCTGAGCCAGGGATGCCTCTGCCTG CATCCCAGAAACTCTGTTCTTCCAGGCTGAGCCCACCATCCCTGCAGAGCCACAGATGCC CCTCACCCTCAGACAGGAAGTTTACCCCTCTGAGCACTTTTACATTTCTCATATCATTTT GTCTTCACAACCGCTCTTCATCTGCTCTCAAGTGAAGAACTGGAAGCAGCAAGATGTGCC CAGGATTCCAGAGTCAGGAACAGAGCCCAGGGCTTTGGCACATGAGGGTGGTTGCAGGAA GGGCTGGTGCCTTCTCTCTGTTGTCCCTAAATGTCCCTCTGCTCAGCGGGGGGCTCCTCC TTTATGGTGGAAAATATGCTTCCAAGCCTCGTCCACTGAGGCGGGTCACTCCAGGACATC CCCAAACTTTCCCTCAACTGCTCTCAGAAAGTCCATTCCCCCAAAAGGGACAGCATCCCG GGTGGGCAGGAAGAGGCCCAGGCCGTGGCTGTCTTGCCAGCCAGCTGCTAACCCGGTAGG CCAATGTGGGGCTCAGCTTAGAGATTGATCAATCTTCCCTTCCATTCCTTCTCTTCAGGT GCTTAACCTGTTCATCGCCCTGCTATTGAACTCTTTCAGTGCTGACAACCTCACAGCCCC GGAGGACGATGGGGAGGTGAACAACCTGCAGGTGGCCCTGGCACGGATCCAGGTCTTTGG CCATCGTACCAAACAGGCTCTTTGCAGCTTCTTCAGCAGGTCCTGCCCATTCCCCCAGCC CAAGGCAGAGCCTGAGCTGGTGGTGAAACTCCCACTCTCCAGCTCCAAGGCTGAGAACCA CATTGCTGCCAACACTGCCAGGGGGAGCTCTGGAGGGCTCCAAGCTCCCAGAGGCCCCAG GGATGAGCACAGTGACTTCATCGCTAATCCGACTGTGTGGGTCTCTGTGCCCATTGCTGA GGGTGAATCTGATCTTGATGACTTGGAGGATGATGGTGGGGAAGATGCTCAGAGCTTCCA GCAGGAAGTGATCCCCAAAGGACAGGTAAGAGTTATCCCCAAGGGACAGCCACAGGCAGT GGGGAGGGGCTTCAGGGCTTGAGTGACAAAGGGCAAAAGGGAAATGCAGAGGAGACCTTG ACATAAAGAAGTTTAAACCATGCCAAGCTTTCCAGAGGAGTTGGCGTTTGAGCAGGGCCT GGAAGAAAAAGTCAGAATTCCCCAGGCAGACAAACAGGGAGTGGGGGGCAGCAGGTGAGC AAAAGGCCACCTTGAGGTGCTTGGGGATTGTCTTTCACACAGCTGTCCCATGCGCTCTGC CCAACCCAGGCTCTGTGCATGAGATGCTAATGAGATTTCTCTAAGAACAGGATGTTGTCC AAGAGCAAGGCTCATCTAATCTTTACCAGCATCCTAAGAAGTCCTGATTCTTATGTGACT CATTTAGCCAGCTTTTCCCCACAAGTGGTATCACAGGGAAATGACACGCTCCTTTTGGCA CCAACTAATTTAAAGCACATTTGAGCCATTTGATAATATATATTTTTAAATACTTGGCAC TATTCCTTACTACCTTAAGCTGGTATATCAACCCTCGCTAACCTCTTCTCAGTAACTCAG GGCATTCACCTCTGCCCAACGTGGGAAGGCAGGGAACCCTGTGAGGCCTCAGGAACATGC AGGGCTGCTGGCCAGGTTACTAAGTGGTCCAGAAAAGCTGTGCTTTGAGTCTGTAGCTCT CCCATAGCCTTCTTCCCTGCAGTCGTCACTTGTGTTCCAGCAGCTGCTGTTATCAGCTGC CATATTTTACATGCCTTTGTCTAGGTAACAGCAGAGAGCATGAACTCTGTGACAGCTGTC TGCCACTCAGAAGCATTTTCAGTTACTTTGGGGCTCTTAGGGAGCTCCAGGTGGCCAAGT TCCCTACAATTTTGACCTCTGGACAAATAACAATGGTCATGGCTAAAATCTATTGAGCCA AGCACTTCGTGCAAAGTGTTTTACAAGCGCTAACATATTTAGTCATCAAAACAGCTGAAT GATGCAGATCTAGCCTTTTCAATTTTGAAGCTGGGAAAAATGAAGATTAGAGAGGTTAAA CAACTTGCCTAAGGTCACACATCTTATAATTAATGGAGCCCAAGGATAAAACATAGGTCT ATCTAGCTATAGTCTTCATGCTCACTTCACACACGTGTATGTATATATGTATGTACATGA GTTGCATATATGAAACTCTTGGCTCTGGCAGCCTGTGATGTGCAAGATCCCTTCAAATTT AGAACTGGTCATCTAAGGGTGGGGCCAGGCACCCAGTTGGGACCCTCTCTCACTAGCAGG AGCAGCTGCAGCAAGTCGAGAGGTGTGGGGACCACCTGACACCCAGGAGCCCAGGCACTG GAACATCTTCTGAGGACCTGGCTCCATCCCTGGGTGAGACGTGGAAAGATGAGTCTGTTC CTCAGGTCCCTGCTGAGGTAGGTATGTCAGCTTCCTGGAGAGGTTGGACAGCCAGCCAGA CACTGAGCAGTGGGCTGGTAGAGGCTGGAGGGTGGCGGTGAAGGTGGGGTATTCCAGACT GCACTCAATCACATAACCTCATTTCTTATCCTGGGAATTTTTAAAGTCCCTCCCAACCTC CGGCTAATCCGCACAATTTCAACTTTCACCATATACCATTGCCACAATCTCACTCCAGGG TGGTTCTAGGTATAGAAAGAAAGAGTGAGACTCCACAAGCTGTGGGACCTAAGATCAGTT CCCAAACCTCTCTGAGCCGCCATTTATTCATCTATGAAACTGAATGGTAATGCCTACTTG GTAGGATTATTTTGAAGCTTAAATGAGATGCTGCTTGTCAAATGTTTAGGTCCTGCCTGA GACAAAGTAAGTGCCCAGGAAATGACAGCCAAGAAAAAAGGAAACGAAAGACAACGCACA AAGAAAGTCAAATATCTTTCAGAGCCAGCAAATAAGAGTTGGAGGTGCTCACAGATGAAG AAGTTTTCCCTTGTGATTCTCCCCCACTTTCTTTTGTCCACATCATTTTCTCTAGGCAAA AGTAGCCTGGGGCTAGGGAGAGGCTCTCTATAGGTGAGTAGTGAAGTGACAGCCTCACAG AGACTGAGCTGGAAGATTAGAGGTTTAGGATTGTCTTATACAAGGTAAAAATAAATGTGG TTTCACTTACAAATCTTGAAGGAAACAAGCTATAGGGAAAGAGGCATCTATAAAAGTTTA GCATCTTTAATAACACTGCCTTCAATTCAAGAATTTAATTTGACACCTAGCCACTGAGTG CTGACTTTGTGACAGGCACTGGGCTCAACAGAGATAAGGAAGTCATAATTCATCCCCAAA GTTATCAAGCCCTTCAGCTTGAAGAATGTCAGCTACTGGCTGTGTGCCCTAGGCAAGGCT GACCACAGCAGGGTAAGATAAGAGGGAGGGCTGGGACGGGATTTGGGGGCTGTGGGTGAA CAGAAGCCGCTTGGTTCCCACTGGGTGAGGGCTGTCACATTCTGCACGTGGATGTCATGC TTCTTCCCAAATGTTCCAAGGACTCCCCTGGCTAGTTGTCCCTACTCAATGTTTTCAGTA CACAACTTCTGGGTGTTGGTTTTTTGTTTTTGTTTTCTTGTCCATGACCCCGGCTAGGCA GGAGGGGCCTGACAGCGACAAATGGGGACCCGCCCCACCATTTGGGTCCATTAGCAAAGC TATCAGGTTTACTAATATGAGAGCAACTTCAACCCCTAATTGAATTCATCTGGAGACATT ATAGGGCTGGAGCTGCCAGGAGGGCAGCAGGCCCCTGCAGCATTACTCCATGATTAAATA TTCAAGCCCAGTGAATGCTGAGAGTCGTTATGGGTGTAATTACGGTGTCTCGGGCCATTG CCAGCACTCCATCAGGGGCCCCGAAGGTTTACAGTCTCACACAGCTAAGCCTCTGGGGCT CCAGGGAGAAGAGCAGTGTTCCAGGCCTCAGCTGGCGAGGCACCAAACATGGGTGCAGTT GAACCTGCAGTGACCGTGTGCAATTCCCCGGGCCCCGCGGAGAGACGAGTTCACTGCCCT GGGCTTCTCCCCTGCAGGGAGTGGACGACACAAGCTCCTCTGAGGGCAGCACGGTGGACT GCCTAGATCCTGAGGAAATCCTGAGGAAGATCCCTGAGCTGGCAGATGACCTGGAAGAAC CAGATGACTGCTTCACAGAAGGTGAAGGGACAGCCACAGGCCCCCTCACACTGATGTTAA TTAGGGCCAGAATCCAGTTGAGCGAACACTCACCTGGGATCCACAGAAGACAAACAAGCT GTGCCCACTTGCTGCCTCACTCCCAGGCTTGAGGGCGCCCGCATCAGACACACATGCCCC AGGTGGCAGCAGCCTGGCCGTGGTATCTGCATCTCATGCCCTATCAGACACTACGGAGAG ACGTGGGCCTTCCTCTTTACCCTCAAAAACCCCTCCGTGAGTTCTAAACCAGCCCTCTTG AGTTGAAAGGGGGAAACGTAGTCACCACCATAAACACCACTGAAAACTCTTGATTTGGAC CATGAAGGACCCAGAGGAAGGCCATGATGTATCTGCTTCCCTCAATCTTTTGTTTAGCTC TCAAAAGCAGAACCTGTGTCTTAGTCATCTTTCCTCTCAGTGTTTAGAACAGTGAGGCAC ATTACATTCTCAATAAATATTTGTGCAATGAATGAATCAAGAGTCCATTCTATAAAGGGA ATTGTGGCCTCAATGCTTTCTAAAAATACAAAGAAAAATTTTTTAAATACTTGAGTTTTT ATTAATAGTTTTATTTGACCCTGGGCTGGGCTGAGCTGGGCTGGACCCAATCCCCATCCC AGCCACGTCGTGGCCCTCAGTTCAGCTTGGGCTTGGAGCATATCCCCCTCAGGCACCCCC AGGCTCATACGCCGTAGCTTGTCTGTTTTCCAGGTATAGGTGGGCAGAAGGACAGGGACC ACTGGTGTCATCTGTAAGAAGGATATGGCTAGGAAAAACATCAGAGTGGGCAAGACTGAT GATGGAGAGGAACACCTGTTGGTCTGGATTGAGGGACTCCCTGGAAGGGACCCCTCTTCA CCCATATTCCAGGAAAGAATTTTTTTCCAAGGGCATGACAAGTGGGTTTCCTAGCAGAAT GGTTTCTCTGCAGCAGGAGCAGAAATTATCCCCTTTGCCCACTGGCCATCACCCACCATC AGTCACCTCTCCCCACAGGATGCATTCGCCACTGTCCCTGCTGCAAACTGGATACCACCA AGAGTCCATGGGATGTGGGCTGGCAGGTGCGCAAGACTTGCTACCGTATCGTGGAGCACA GCTGGTTTGAGAGCTTCATCATCTTCATGATCCTGCTCAGCAGTGGATCTCTGGTAAGGG GAGGCATAGTCCCTGCCCCCAAATCCTCCAGATAGGAATCTCCTACAATCCAAAGGCCCT GGAGCTGCATCAGTGGGTTCTCCTGACTACTGAGCTGAAAGTCCCCAACCTTGTCACTAG AAGGAGAGCTGCAGCCCAGGGCGAGGGCCCCTCTTAGCAGCAGAGCATGGGAGGTGTTCA AGGTGCCCGTGCCATGGCCTGCCAAACCTGGCTGCAAGCATTTGAGTGAGTGAGTGTATG CCTCCCACTATATAGATGGGGAAACTGACACTCAAAATGTTTAAAAGGCTTGTCCAAGGT CACCCAACTTGTCAGTAGCAAAACCCAGCTTTGCCCTTATTTAATCCCGAAGCCCCTGGC TTCCCACCCTCTGACCTCCCCAACACACGGCCTTCTCTGAAGGCCTACAATACAAAAAGG ACTATCTTTCCTTATCAAACCACTTCCAAATGCTACTAATTACTCCTTTAAAGCTCACAG AAAATCATTACAATAGCACTTCTCAAAATTTAATCTCCCAGGAGCTTGTTCAAATGCAGA TTCTCATTCCATAAATCTGTGGCAGGGCCTGAGAATTTGCATTTCTAACAAGCTCCCAGG TGATGCTTGCACTGCCCTCAGTGACCCACACTTTGAATAACAAGGCCTTATGGGAATAGA GATTCAGAGTTGGGAGACATCTCTAAGGCCCTGGCTGAATACTGGGCTTTGTAGAGCAAA GAAGGAAGGTTCTCTCTGTACTGTGGCTTAAGCTCCTTACACAATCTCAACTACTCCGCC AATAGACCAAGGTAGACATAGACACAGGAAGGAACTTAACATCCTCAGTTGTTTGCATTT TCTAAATGAGGAAGTCAAAGCCCAAAGGCATGAGGGGCTTCAGGTAATTAGTTGAAGGGC TGGGAATAGAGAGTTCAGGGTGGCAGCTAATTAGTTGAAGGGCTAGGACTACAGTCCCGC TCTTCCGATTCACGGCTGCAGTGTTTTCACTCATCTTGCGCCTCATGTGTCATTTACTCT CTAAAAACAACCTACTAAATATCTAGGCCTCCATCTAAATGCCAGATTGTCCAGGAAGGG TGACAAAACTGCTCGGTGCATGGCCTCAGCTTTAGGGAGGAGCACAGGTGCGGGACGAGG CAGGATCTGAGAGGTGGGAGGAAAACTAGGGTGGCAATGACGCAAAGCCAAGGGAGAAGA GAGTTTCGGGGCAGGGACTGGTCTGCCATCCTGGGTGGAGGGGGAACTGTGTGACAGCGG CTGCAGGTGCCCAGTGCCCTGGCACAGGGCAGTGATTGTCCACACGGACCTCAGCACAGT GTAGAGGCCAAATTGCAGGGGCAAGAGAGGGCTGGGCAGTGCTTTCTCCACACCTATGCT CTTCTCCTTCCAGTCCTGCTGGATTAAAGTAATAACCACATGTGCTGTTGGCCAGGTGCT CTCCTCACTACGTCATTGGCTTGTGGGCCTTCTCTCCTCATTTTGCTTCTCTGCTCTCCC TTGCTCCCTCCCTTCTCTCTCTAGCCCCCTTCTCTTCACACCCCGGTCCACCCCCCACAT TCCCAGGAGGAAGCTCAGACCTGCAGCACTGCGCCAGCCCAGCCCGCCCCCTGCAGCTCT CCCTTCCCTCCCATCTCCTTCCCCTGGGACTGAACAGGCTGGCACCAAATTTAGCCTTCT TTAGGCTTTCACTTTGAGTACCTTTTAAAAATAACATGCATTTTCATCTGACAAAGTAAA GTACCAAAAAAATACTTGGAAATTATAGAAAAAGGTAAAGAAAAAAAAATCATCCATTTC CCTATCACTCAAAGAGAAACTCACTTGAAAACATGCTGGTATACCTCTTTCTCATCTTGC TTATGCATAAAATATACACATATTACAAAAATAAAACCACATTTATGCATTCTATAACCT CAGTACATTTTTCTCAGTAAATAGTCTCATATTATCTCATTTAATGGCTATAACATATTC CTTTATAATTTACTCAACCAATCTCTATGTTTACTTCTAATACACATTGAATATTCCCTG TGTGTACTCCCTTAGAGACGTTGGATAATCTCCCATCTTAGTCCTCGTCCTAGACACAGA ATCGGAAGCTGAGAAAAGATGTGGGTTCAGTTGAAATCCGGCTTCAGCTGATCCCATGGA AAGCCCTGAAGCACAAAGGACACCAAAGAGCTGTCCCACCTTGAGGCAAAGAAGACAGCC TTGTAAATCCCCCTGTCACTGTCCCTGGCTGAGGGCTGCCCCAGCGGGGAGGATAAGCAT TTCCCAAGGTGGACACAGTTTCCTCCAGCCCAGCGAAGCCATCAGGAAGGGGGTGTCTGC ATCCATGAACTATTAGCAGCCGACACTCCCAGCAGATGAAGGATAGGGGCACTGGCCCCT TAAAGAGGACCTGAGTGGGCACCAACAGCCTCCAGTGTCCTTAGGATTAGTGCTTGAAGG AGGAATTGCTAGGTTCCTGAAACCTCTGAGTCACATTGCCAATTTGGCTTCACACAGGAC TGGCCAGGTTTTCACTCCTGCCAGTCACTTGTAAGAGTGGCCGGGCGCGGTGGCTCACGC CTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCGGATCATGAGGTCAGGAGATCGAGA CCATCCCGGCTAAAACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGC GTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGA ACCTGGGAGGCAGAGCTTGCAGTGAGCCGAGATCCCGCTACTGCACTCCAGCCTGGGCGA CAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAGAGTGGCAGCCCCCCACAGCCACAGCATCCTAGGGTTTCCCCTATTCTTTTCCATCT CCACCAGTCTGATAAAAGAAAAACAGCATCTTCTTTTAATTTGAATTTCCTTGATTACCA GTGAGATAGAACATTCTTTTTCAGAGGTGTGTTGGCGATTCTTACTTTCATTTTTGTAAA CTGCCTGTGAACATCCGTTGATGCTTTTATTTTTAAACAAAGTCACTGTTAGCATCCCTT TGGTTCACTTCCAAAATCCAATTTGAGAAGGTTTTCTTTCTCCCTTGCATTCAGCATCCC TGCAAGTATCAGCAGGGCTGTTTGTCCCCATTGGCCCTGAAGAAGTCATGCTTTGGATCT CACAGTACCTCCTCCACTATCCCACTTTACCTCCCCACAGCAAACAAAATGCTACTGGCA AGCTGACTACCAAATCCCCTTCCAGGGACTGTGGTCAGGGCTGGCTGGGTCCTTCCCTAT CAGTCTGGGCCCCAGTGCTGTGGGTCCAGGCAGCCTGCCATTCTGCATCCTTTCTTCCTT CCAGGCCTTTGAAGACTATTACCTGGACCAGAAGCCCACGGTGAAAGCTTTGCTGGAGTA CACTGACAGGGTCTTCACCTTTATCTTTGTGTTCGAGATGCTGCTTAAGTGGGTGGCCTA TGGCTTCAAAAAGTACTTCACCAATGCCTGGTGCTGGCTGGACTTCCTCATTGTGAATGT GAGTGGCTCAGTGGGAACACGTGGGGTTTGGGGTCCTCCCCTCTGACTCCTGGCTACAGC TAGGGCAAAGGAAGCTCAGAGACCTGGCCCTCAAGGGCTACTCCAAAGAAAAGTTTTGAG CATTTAGTGAGGAGTCTGAGAATAGCCCTGACACCCATGAGTGGTAAAGTCCAGGCAGTA TGTGACTGTGACACAATAGTGACTGTTAGGTGAGTTATTAGGTCTCCATTCAGAAAGATC CCTGCAATCCTCTGCATTTTTACATCCAAAGTGAGATGACACATGCAGCTCTCAGAGGTC TTATGGCCCTGCATCCATAGATGGGTCTTCTGGACCTTGGGGAGCCATGAACTGTGAGCA CAGAGAATCTTCCCACCTGCGGTTCTGTGGTCCTCACACCTGGCTGCACTTGAGAATCAC CTGGGGAGCTATGAAATATGCAGTGACCGACCACCACTACCCCAGAGATTCTGATCTTAT TGGTCTTGGGTGGTGCCCAGGCACCTGTATTTTATAAATGCTCCCAGGCATTTCCAAGGT GCAGCCATGGCTTAGAACCACTGAGCTTATACTGCTGCCTCCTCCCCAGTGGCATTGAAA AGAACAGAGGGCGCATTTGGAGAGGTAATGAGCTGGAGTTCATGTGTTCACACTCATGGT AACCTGTGGCCAGTAATCCCTGCATTAGAGAATTGACTGTGTGCTTCTCACACATCAGAC ATGTGATTCCTCAGTCATCTCTTCCTCATCTTAACCACCACATTGCACTATCACTGTCTA GGCCACTACCTTCTTTGCTTTGCAGCATGGTTAACTGAGTTATCTGTTCACTGCCGCATA GCTGAGGGTTTCTGCAAGCAGGGTGAGGCATGTGCTGTGTTGGTCTCTGCCTGCCCCACT CAAGCAGCTGCCAGCAGCAAGGTCTGCCCAGCAGAGGTGTCAGGAAATGTGTCTGTATTT GGTGCTTGCCTCCTGAAGCTGGATGAGGTTCAGCACAGAACATTCTGTCCAGCCTCCTGT GCTGGCAGGTCCTCCATAGCAGGCCTGTGCATTCAATTCTCTTCTTCCCTGATGCCAATG CACTAGCAGCTTCAGGCCACTGTACCACAAGGATTAGCTGTGAGGCCTGAAATCTGAGTC ATGGGTGATAATTCTGTGCATATGAAATAATTATTCATAAATGAAAGGAAAATTATTTAT TCCGGTGTCCCACCCCTTTGGGGGCACAGTCACTCCTGCTTATGTTGATCTCACCAGTCT CAGTCCTAATCACCTTAAACGGTCATGCACTCATTTCATCCATCTTCCTACTGATTCTTC CTTCCAACCATCCATCCTTCCTTCCATACATTTCACCTTTTGTATCCAACCATTCAGTCT TCCTTTTTCCTTCCTCCCAAACATCCATCCATCTTTATTTCTTCCAGCTGTACATCTTCA TCAGTCCTCCCCTTCCATCCATCCATTCCCATTCTTCTTTCCCTCTTCTCTTCTGCCCAA TCTTTTATTCTAAACTTCCTTCCATCTATTCATCATCCTTAACCCTTATATCTTTCTATC TTTTGATTCATTCTTCTATCCAACTGTCCAATCATCTAACATTCTGTTCACTCATTCACG CATCTTCCATCCAAGCATGCTTCTACTCACTCATCCTTCTATCCACTCATTCTTCTATCT TTCCATTCTTGTTTCAACCTATCCATCTATTCTTCCTTTCTTCCACTTATCTGTTCATTC ATCCCTTTCCCTTCCATTCATCATCCATTCATCCATCCTATCAACCTTCCAGTCATTCAC TAATTTATTCCATTCATCTATTCTGTCCAGCTCCCCATCTTTCCATTCATGCATTAGTCA TTTTACCCAGCCATCCATCCTTCTCTTATCCACCTATCTCATCTATCCATTCACTATTCC CTGCTTTCATACTTTTCCTTTCTTGTTTGTTTCTCCCATCCTTCCATTCAACTATCTATC CTATCCATCTTTTTGTGTCTGTTATCTTTTAAAATTTTAAAAAATATATTTTATTGTATA TATTTGTATTAGTCTATTTTCATGCTGCTGATAAAGACATACCTAAGACTGGGCAATTTA TAAAAGAAAGAGGTTTAATTGGACTTACAGCTTCACATGGCTGGGGAAGCCTTACAATCA TGGCAAAAGGAAAGGAGGTGCGAGTCACGTCTTATGTGGATGGCAGCAGGCAAAGAGAGA GGTTGGGCAGGGAAACTCCCCCTTATAATACCGTCACATCTCATGAAACTTATTCACTAT CATGAGAACAGCATGGGAAAGACCTGCTCCCATGATTCACTTACCTCCCACAGGGTCCCT CCCCTCCCACAACACATGGGAATTCAAGATAAGATTTGGGTGGGGACACAGCCAAACCAT ATCAATTTTTAAGGGATACAAAATGATGTTATAAGATATATATATATATAAATGATTACT ATAGTGGAACATATTAACATATCCATCATCTCACATAGTTACCCATTTCTCCCCCTGTGG CAAGAGCAGCTATAATTACTCAGCGAGCAAAAATACTGAAAACAATATGCTATTATTAAC TATAGTATTCATGTTATACATTAAATCTATCCTATTTATCATTGTATCCTTTAATTCATC CCTCTATGCAATCACCCTTCAACTTTTCATCCAGCTGTACTTCTTTCTTTCTTTCCTTCT AGCCATCCCAATGATCCTATTTTTCTTCCATTTGCTATACATCTAACTATTGAATACAGG ATTGTCTTATGAAGTGCTTTGTAAGAAACAGAAGCACAGAATAAATACTCACTATCTCCA GGATCTTACACTTTAGTCTTTCAGCAGACAAGACACACATGGAAATAAATGTATTATAAG GGTGAATGTGTTGAGCACTGCTGGGGGCAATTAGTATCTTGGAGACACAGAGACATGAGC CTTCTTCCACTCTTGGGGTGGGGTGGTTGAGAATTCTTCCTATGGGAGGTGGCATTAGAT GTGGGGCATAGAAATGAGCAGGACCAAGGAGCTGCGAGAGGACTGGGGAAGGCCCGTCTT AGCTAAAGGCTGACCTTGAACAAAGGCACAGACACAAGTATGCTCAGGGAACAATTTGAT TTGGGTAGAATGTGTGGGTGAAATATGACTGGAAAAGTAGGTTGTGACCATATGACACAG GCTACAGAGTATGGAGTTTATCCTGAGGGTGATAAAGTACCAGTGATGTTTGGGAGTAAG AGAAAGACATGATCATAATCAGATTTGCAGTGGAGAATAGATGCATGGTGCAGTACTAGG GCCTGGAGACTACTTGGGAGGCTAGTGGGCTGAGCAGTGGGAATGGAGGTGGGCTTGGGT GGACAGACTTTGCTGGAGAGGCACAGCACCTGTCTGCCCCAGCTGCAGATTATCCATGTG AGGCAGACTTCCCAGGGGGCTGAGCAAAAGACTGAATAGGGTTCAGCATGGAACTGGCCC CAAACCTCCCTACCCTCATGCACCCAGGCAGCTGGGTCTCACCTGCTGATGTCTGGAGCC TTGCATGAATGAAGGAAGAGGGTCCCTTCCATTCCGGTTCTTTGTCCTGGTGCAATAAGC CACTCATCCAAAGAATGGACATTGCTTGGGGCTCTGCCACTCAAGTATAAAGCCCTTCTT TTCTGAAGCTCCTGTGTGCATCCCACCTAACTGCCAGGATAAGCTGAGCCTGAATTCAGC CCACCATCTGTGGTGTGGCTAAGATCTCACAGGACAGGGTCATAGCCCGTATCTATGACA TCTCCCTGGCACCTTCAAAGTTCCTAGCAGGGGAACTTTTTTCATGTAAGTTTCTGTCCT TTGCAGTCTTTGGACCACCACTTAAAGAAAACTGACAGACCAAAAGGGGCTCACAAGTCA GTCATTATATCTCAGATATCTATTGAAGCTAGATCTCATGGACCCATACCAGAAACTTTT TGAAGAACTGCAGAGCTATCAGAACTTTGGAATTCTCTTAGTCTGACTTTTTCATTTTAC AAAGGGTGAAAGTACCCTGGACTAGCCAGGCACTAACCCAAGCTCACAAAAAATCAGGGT TCAAATGGGAATTAGACCCCACCCAAACCACTATTCTTTCCACCCCACTTCTCTGAGCAA ATATAATTCAGATTACTATATTCCTCAGAATAGACTCTCAGCATAGCCCCTAGGAGGGAA CATCATGTCTCCAAGTCTCTGAAAATCAGTTTCAAGGTTGTTGAGGCCTGACTGCTGCCC CTAGAAGGAAAAGGAAGAGTTGATTTAATACACTGGAAGCTTGGCCACCTACGTACCAGA GGTTGGTGATGTTAGAGACTGAGTTTTTCTTGCCCAGATTGTTTAAGAGAGTGGTGAATA AAATTTTTAATAAAAGGGAAGAGACAAAACTCTTTCTAGACCTCTCATTGGAGTTCCGAA TAAGGTGGGAGCTAGGATGATGGCTTAGACGTGTAGGAATGGGAGGCTGGGCAGGGAGTT CCTGTCTCCTTCCACGAGGACTGCCTTTCTGGAAACCTAGAGTGATGTTTCTGCCCTCTC CTGCACTCAGATCTCACTGATAAGTCTCACAGCGAAGATTCTGGAATATTCTGAAGTGGC TCCCATCAAAGCCCTTCGAACCCTTCGCGCTCTGCGGCCACTGCGGGCTCTTTCTCGATT TGAAGGCATGCGGGTAAGTCTGATCTCAAAACTTCTCTCATCTGGCCAGGCACGGTGGTT CATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCGGATCACCTGAGGTTAGGAG TTCAAAACCACCCTGGCCAACATGCCGAAACCCTGTCTCTGCTAAAAGTACAAAAATCAC TACCACCACGAGGTGTGGTGGTAGTGAGCCGAGATTGTGCCATTATACTCCAGCCTGGGT GACAGAGCGAGACTCCGCCTCAAAAAACAAACAAAAAACTTCTCTGGTCTGCAGGTTTTG GTAACCCTTCCCCCTTTATGCCCTCCATCCCACCAACCCTGAGCTCCGGGGCCCCACCTC CAAAGGTAGGCTCTGAGTCTTCACTTCCTGCTCTGACTTTGGTGGTGCTGCATGTCCTAA CTCTCTTAGTTCTCTTTCAAGTCTTTTTGTGGGCCCATCTTCCCTTGTCACCCATAAATA TTACAGCCACCAGGACCCAGACCTCACCCCCTTTGCTCTTAGTGAAGTAATCCTGAATCA TCGCTCCACAAGCATCTATTATCTCAATCACCCAACTCTGCCTCTTATTGCCAACATCTC CAGAACATCCCAAGTTCTCTGTCTTTCCATGACAAATGAGTCTTTCCTCTAATACTCCAT GAGTGACATCATCAGCTCATTACCTCAGCTGCCACCCACTGGAATGCTTCTTCTCTCCTA CTTAATGCAATCATCAGGTCCTGTAAATTTTTCCCTTCCATCCACCCCTTTCCTTATCCA CCACTGTCAGCCTCCACCTTGCCCTAACTGCCTCCGTCAGGACAGAATGCTCACAGCCAG AGCCTTGTCTTAGAGATTGCTGATTGACACCCACCTCTTCCTCATAATTACAAATGGCTC CCCAGCAGAAAGTAAGGTTTTGCTTTGCTCCACTATCATTGGCTGGAAGTAGTCATCATA GCTTCAAAAACAAAAGTGGGCTTTTGAAATTAACTTGTACCTTTACAAACACTAGGACCC GAAGATTGAAATAGTTTGAAATTTATTCTATTTTGTATCTAATAGATTCTCATTTGCTTA TTTACAAATAAGCTATTTTGTGAATATGGATTTAGGTTTTCACTGGTTCTTGACCACGGC ATACTTTAAATTGTATTTAAATTGTGTTTGTGTTTTATGTATGGTATTTAAGGAGAATAA GTCAAGGTTCTAGAAGTCTGTATGTGCAAGTATAATACACACACTCACACTTCTAAATGA GTGCAGTATGCTTGTAAACTCATCCATCTTCTCACTGCAACCCACCCTGAATGAGGTTAA GGGAAGAAGGTCTAAATCCATCTTCAGCTGCCCAATGTCTCTCAGTCTTCTGAGTCTGAA TTATTGCACTAATTAACCCACTTCTCTTGCTTCTTCCAAAGGCCACTGGCTCATGAAGCA GCCTGAATGAGTATAGAAAGAGATAGAATCCAGCAGAGGGGGTGGGGCATTGGGAGGTTC CAGGTCTTTCTCTATCTCCCACCAGGTGGTGGTGGATGCCCTGGTGGGCGCCATCCCATC CATCATGAATGTCCTCCTCGTCTGCCTCATCTTCTGGCTCATCTTCAGCATCATGGGTGT GAACCTCTTCGCAGGGAAGTTTTGGAGGTGCATCAACTATACCGATGGAGAGTTTTCCCT TGTACCTTTGTCGATTGTGAATAACAAGTCTGACTGCAAGATTCAAAACTCCACTGGCAG CTTCTTCTGGGTCAATGTGAAAGTCAACTTTGATAATGTTGCAATGGGTTACCTTGCACT TCTGCAGGTGGTGAGTCAACCATGAGATCAACCATATCTCTTGCTTTGGCCGCTCAATAA AATGGAGAGTCTAGGTTCCCAGAAGATGCTATGTGGGGACTTTGCAAGGAGTAGCACTCA GTGTGAACTGAGGTCATTCTCTATTCCTACAGACTACACCATGAGGCTAGAGGGCCTTCC TAGTGAATATGATATCACAGTGGTGCAATTACACAAGTGTGGGCTAGTAGAATAGCATAA AGGGATGGGCCTGCCATGGGATTATCAGGTGGTGAGTTAAAACACAGAACCTGTACAAAA TAGACACGGCATAGCCACATGTTCAGTCTTCAACTTCACATCATTTCCTAAATGGTAATA AATTATGTCTCATTAGGTCTATTGTAATGATATATAATGTTGGTGATAATGACAGTCAAA CACATTCATCCTGTCTTTCTTTCTTTCATCATCCAGTCAATCACTCATTTCTTGCCTTCA TCTACCTGTTCATTCAGTCCTCTGTTCATTCAAGTGTTAATACAATTACTTATACATTTG TCATGCAGTTACTCTTGAGTGTCTACATTCCACCATTGGTTCTTGCATTTATTCAACAAA TAATCTCTGAGTAGCTCTGGTATGCCAGGCATGGTACTACTCAGCCCTGGGAGATACAGT CAGAACTGGGCTTGGACACTGATGCTGTATGAGAAACAGACTAATAGGTGAATTAAAACA GCTCATTAAGTGCTGTAACAGGCCTAGGAGTGTAAGAACTAAGATGTCTTGCATGCTCTG AACATGCTGGAAGGCACACCCAGTTTCTAGACCTCAAGAATGCTAAAGCTGAAGGCAGGA ACATGGAGGTCTTCTATTCTCAACTTCTCCATCTGGAAGATATACCATTCTTACATCATG TGCATAAGGTGGGAAAGTGGCTGAGAAAATGGCAGCCCTGAGAGGGGAAACCTTAGGCAA GTGCACAGAGCAGACTTGTAGCAATTGGCACTAGAACCAGACACCTAGACACTCAGTCCT CAGTCCATACTACATTCTGAAAGGCTCTGTGTCTTTAATGCTACTCTATTCTTAAACAGC CACATGTGGGATTACTTTAAATCATCATATGAGATTAGTCAAGTAACCCAAGGGAACTTT TCCTTTCATTCTTTCTGCACAAGCCCTTTCCAACACAGGACTCTCTCCTCTCTCACTGGG CCACCATGGCTTGGGAAGGGTCCAGTATAGATGGATGGGCAGACACTGAGTGGCCTCTGC TCCTTGTCTCCAGGCAACCTTTAAAGGCTGGATGGACATTATGTATGCAGCTGTTGATTC CCGGGAGGTGAGTCTCAGCCCACTGTCCCTACAGCCTTCCCCTCTGTGCATTCTACCCAT ATCAGTCTGCATTTGAAATGAAGTGACGATGACCCTGGTACTGGAAGAGGCGATCACCCA GCAACCTCACACTGTCCCTCCTTGCAGCAGTGTCCAGCTGGCTCTCTGTTGAGTGTTTCA GCCACCGAGGCTTTCCCCCTTTCATCTACACTCCCCAGGCTTCTCGAGGTTTTCCTCTGT TCACTGTTGAGGAAAGCATCAGATAACCTCTAAGAGACAGTTCATGTGAGCCACTCCCTG GACAAGGGTGGGAGCAAAGCAAGGGTAGTAGCTAAATCAAGGTGCATAGGGAGGAATGAG GTAGAATCTCAGGAACTATCATAAAACCCAGGATGGGCCAGGCGCAGTGGTTCATGCCTG TAATCCTAGCACTTTGGGAGGCCGAGGTGGGCAGATTGCCTGAGCTCAGGAGACCAGCCT GGGCAATACGGTGAAACCCCATCTCTACTAAAATATGAAAGAAATTAGCAGGGCATGGCG GCTTGTGCCTGTAGTCCCAGCTGCTCGGGAGACTGAGACAGGAGAATCTCTTGAACCCGG GAGGCAGAAGTTGCAGTGAGCTGAGATTGCGTCACTGCACTCCAGCTTGGGCGAGACAGC GAGACTCAATCTCAAAAAAAAAGAAAAGAAAAGAAAAAGCCAGGCTGGGGAGACACTATT GGGGAATGACCACTTCCTGCTGTGGCAGGCACTGTCAATTCCCTGCCACCTCTTCCCTTA GCCCACTTCTGATTTCACCTGCAGGCTGCACTTCTGATTCTCTGTATAAAGACTCCAGGG CTTAGGAGCTTGTGCAGCAACTTCTCCCCCAAGTACCAGGGATTTAATGTCCCTAGGGGC AACCCTTACCAATAAGTTTTGGGAGTCTGAGGATAAATACTCCAGTTTCCCTGTCCCTTG GTGATATAATCCTGCATGTGCTCTACACAGTTACTCAGAGGACCCCAGTAGGATTGCGCT TCAGTTGCCCACAATGATGCCCTGCTTAACAACATCCTCTTCACAGGCTCTTCTCCCATT CCCGCTTATTCTTCCCCACTCCCTCATTCCTGCTTCCTTGGATCACCTCCCAGATAAATC ACCTGCACCCAGGTCCATGTCTTAGGCTTGGCTTTCAGAGTGAACCTGAACTAAGACACC CACCCTCCTTCCCACTCTCCCTTGTCTGGTCACAGGTCAACATGCAACCCAAGTGGGAGG ACAACGTGTACATGTATTTGTACTTTGTCATCTTCATCATTTTTGGAGGCTTCTTCACAC TGAATCTCTTTGTTGGGGTCATAATTGACAACTTCAATCAACAGAAAAAAAAGATAAGTG GTGCTCAGGAGTTTCTGGTTTCTGCTGTAGTGGTAAGCCTTATATTTCCTGTTCCCTGCC CAGCTCTGGCCTCCTGACAAAAGCCAACACTACTCACAGCCCATCACTTTAATATCAGTG CTAACCCTTGCCCTGTGATTGCTGTTGCAGGAAGAGTTGCATTTCTCTCTGTCAGCAATG CCCCACTAACATCCTCACTCAGGATTCACATCTCAAAGGGATCTTGGTCTTCCCAACTGG AACCAGGTGTGTTAGATGTGTTCCAAGTGAGCCCTGGGAGAATACAAGCAGGGAAAGAAA GGGATCTAGAACTTGCTGACCTGTTGGGGGATATGATCCCCACAAAGGACTGAGTCAGGG CACCTCAGGAAAGGCCATGGCCCAGCACTGGCAGAATCCCAACCCTCCTTGGGATGGGGA GGAAGGCTTGTGCCTCCAAGGGTGCCGACTCTGATTAATAGGAATCGAATCCCAGATAAC CTCTCATGTTTATAAGAAAATGTCAGGGCAATAAAGGTATAAAGTATTTTCCTATTATCC TTTCATTTAATGCCTTCCATACCCCTGAGGTAGATAGGCGCTGTTCTAGATTTGCGTAAC TGAGATCCCGAGAGAAAAGGGGCTACATGGATAAGTCACAGAGTACATGCAAACCTGGGT TTGCATTCCTGACTCAACTACTTGTGGATGGCTCATGCTGGCAATATTTTAAACCCCTCT GAGTCTCAGTTTTCTCATTCATTCAATGGGGCTAATAATACCTACTCTCTGGATTGTGGT GAGGGGTAAACAAAATCCTGAACAAAGATCACTGAGCATAAGACAACTTTGGTGGTGCAG TTTCTGGCACTGTAACAGCAGAGGGGAGTGTGACTGGCAGCGCCTCAAGAGCACCAGATG CTGTTGCTATTAACAATTTAAAATATATACCTACTAGGGCAGGCATTCCACTTACAGGAA ATCCTTGCCCAGGGTTCTGAGCATAAGAAGAGGACAGGATAGGTCTGGGACAAGACAGTG CTATTTAGTACCCTGGGGAGGTTTGAGATTATTGAAGCAATCATTTCTCTCATTTTACAG ATGAGAAAACTGAGGTCAAGAAACAGATTACAGAGGGAGATATTAGGTAACGGAAGGAGA TGAGCTAGTGACCAGGCTGGGGTCACTGAGAAACTGCATCTACTGAATCCTTGGTCAGAG TTCTCAGCATGACCTCACCTGGAGCTCACCTACCCACTGCCCTTGCCCTTTCCTTCTCTC CCCCCATAAAGATGGTCCTCCCAGGAAGCCCTTTGTGTCCACAGATCTTCTCAGATCTCC TTTCAAATTCCACAGATCTCAGCCCAGTTTCCCAGCATTTGCAGGGACTAACTTACATAA ACATGTTATTAAAATAGGTGGCTTTCAAATTATGTCAGGAAGCTAAAGGTGAGAGTTTGA ATGAAGAAAATCCAAAGGGCCCCTAAAGAAAAACTAATACCCAATCCTAAGGGAGGAAAA CATCTTCGTTGGTGCAGTTTCTGGCACTGTAACAGCAGAGGGCAGTGTGACTGGCAGGGC CTCAAGAGAACCAGAGGTCCCTCTGAGACCAAGGACCCAAGGATAAGGCAGTAATTGTCC TCATCTCCTCCTCTCCAGAAAAGATTTGAGGGCTTCTGTCCCTCCCAGAGTCTTTGCTTC AAGGTCCTCAGGAAATCTCTTGCCCCCTGGGAGGGACTGTGACTCTAGAAGCCCAAAGAA TTTTACTGGAATGAACAGGCTAGATCCAAGGCTCAGATTTGAACCAACATGTACCTTCTC CTTGAGTCCTGTTTACCAACCCTCCCCACCCTTTATCCAGCATCTCTGGATCCCTGGAGA ACAGAAACTGGCTTTGCTTTAGAGACCCAGGCAGGGAGTCAGCAATGGAACTCAGTCCCA CCGTTCCGACACTGGATGTGGCAAGACACTCCTTGATGCTGGGCTTGGTTTGTCTATCTG CAGAGGAGGGGAGAAGGTTGATATGATCCTTTCTCTCCTGCCTGTTCTGATTTTGGTATC CGTAGGGGGTTCCTCTTGCTTTAGTAACAGTGTGGCCCTCTCTGCACTTAGGGGGCCAGG ACATCTTCATGACAGAGGAGCAGAAGAAATACTACAATGCCATGAAGAAGTTGGGCTCCA AGAAGCCCCAGAAGCCCATCCCACGGCCCCTGGTGAGCCCCAGAGGTTTCCTCTAGCACA GCCTGTCTGTGAATCCAACATGACAGTCTATGCCTGTGTCAGGCAGGGGCCTGAGGGAGT GCTGTTTATGGAAGAGAAGATCTGGGTTGACGTTGGGATGAGGGTGATGAGGGTTCCTAG GCCCAAGGAATGAGAAGTTCAAAAGAAGCAACTCTGGTCTTCTGGAGTAAACATACCTTT TACAGCTATTTGAAATGAATCTGTTGGGGGAAGCACTGAGTTTCTCCAACAGTTTTAGAG TTCAAGTACAGGATTGGAGGCAGGTTAAGCTAAGTGACAGTAAGGGTAGTTAGAACCCAG GGATTAAACTTGTGTGGTTATGTATCATGGTCTAGGTCGCAGTAGATCTCCAGTATCACC CTATATTCTACCCCACGCCTACAGGTATAGAGAGGAAATGACAACAACCCAACTCCCCTG TCAGGATCCAAGATGCAGTCATGGTAGTGGTCAAGTGGCCCAGTAGGAACCAAAAGTTAT CCCACTCGAGTTTAAAGATTGGGCAAAAATTAGGGCCAGACTCTAGTTTCACTCCTTTTT TCATAAAGATCTCAAGAGACATGGCTTTGCGTTCCTCCATTAAATGTACTACCCATACCA CACATGATATCACCTAAGTTCTTCTCAGTCTTCTGCCAGAAGATAGAAAACATCAAGCCT CTGAGAGGCCCTAAGATAGCACTTTTTTGGGAATGGATGAACAGGTTTGGGGGTGAAGAT GTTGATGTGTCAGAAATTATTGTTGATCGTCTTCAAAGAATTTCTGAGCCATCCACTCCA GTGAATGGAATTGGCCCTGTGTCCTTCTACATAAACTTTCTGTGGCTAAGATGCAGCATT AGTGAAGAAAATCACAGGTATTGCTGATGACACAATTTCATTAGCCCTTCCCCACACAAG GGAAAAGTCCAAAGGACATTTAACAAAGGAACAAGCCTTTCCCTAAATGAATTATGGAGA CATGCATTCTGTTAGCCAAGCTATTAGATTTTTTTAAATCAAGGTATAGGATTTGCATAA TTTACTTCAAACTTTGTCAGAACAAAGTGAAAAACATTTACAAGAGAAAAAAGTGATGAC TTTTGACACTGTATTTTGAAATAGTGGTCATGAATTTATTACTAGTCTAGATTCTGATAA GAAGTATTACAATGGGGAGTGGGGAGTGATAGGAGGTTCAGACCTCTGTAGGGGCACAGC TTTCTTCCTAAATCACCCATACAATCAAGATAACTGGATTTGAGTGAACCTGAGCAAAAG TCCTTAGGCTCATTGAGTATCCCTCCATCAACAAGTTCAGTGGAGAGGAGCAGGCTCAAA ATTAAAATTAGGTGAAAGGCTGAGTCCCAACCACATTCAAGTCATGGGTAATGAGAGGAG GACCCTAAGACTTACTGAGAAGCAAGCAGCTGAGTCCACACTGGCCTTTACTTGTGGCTG GAGTAAGATTGAATCCACATTTGAGCCAGATGCCAAGGAGGTGTGTGCTAGGGACAACCC TTGGCAAAAACATGAAAGTTTTTGGCACCTTCCCCCACATACTACTCAATCTCTCTTTGA CATCCCAGCTTCTTCCCTGTCATCACCCAACCCCATAGGCTGCCAAAGAAAGCTTCCTCC CCTCAAGGAAGACAACATTCTTAGGACAAGATAAATCATATTTCAAAAAGGGACCCTGGA GAGGGCATCTACCTACAAAGGCATGACCCTAATGTTGAAATTCCAGGCAGTAAATAGGAG TGGGAGACGGAGTTGATGGTCAGGCAGGAAGACCTGCAACAAAATCACCATTTCTCCTAA TTAAGCTGCAACTAAGATCCTCCGAGGATGGCACAGGGGGAGACCTCCACATGTGGAGGG GAAGTGAAACCTCATAACCAATTCTGTTCCTGAGATTCTCCATCCTTTGAATGGAAGAAG AGTGGATTAGTAATTCACTCTCAACCTTTTGCTAGCCTTAGTGGAGGAACGTGGCTCTCC CCTTAATATTCTGGCCCAAGCAAGGTAAACACTCTTTCCTTTGTTCCCTAATTTCGCATA GCTGAGGCAAGGCTAGAACATTTAAACAAAACTTTGAAGTGGCAGGGATGCCAACAGAGA AATCAGCACAAAGCAGAGTCCAAGCAGCACCTGCAGATGGCTGCACTCCAGGACCCATAG GTCTTGGACCATCCTAGCTCCTGACCAGCATCCAAATGGCAGGCAACATCTTTGACACCC ACATGCCCAGTGGCACAAGCCGGATCATCTTCCCTCTTCAAAGTCTAGAACCAAGAAAGA AATCCAAGGCAAAAGGAAATTCCCACTCCCAACTAAGACCCTTCTTCCCAGGCCCAGCTT GCTTTTGGCCAGATTATTGGCCCTGGAATGTCCACCTTCTCCCCTTAGGAGCCCCAAAGG TCAAGCTGGTCATACCAGCAAAGCACTGACACAGGATTTCTGCCCCATCAGTGCATGGGT GCCATTTCATGACAGAGCTTACCTGGGACCACATAGGAGCCTCACACACCTCCTCTCACC TCTGATGATGCTCAGGCTCTAAATCTTGTTTCTTCCATAGGAAAAGTTAAGGATTCCACA AGACTGGGAAATTTTATTCTTTAACATTCCCTGCTACAGAAATCAATAAAAGCAGCTCTC ACATACTCCACTTTATAGAAATATAATAGGCTCTAGAGAATATCACATAATCAATCACCC ACTTCATCGTCTCTGAGAGCACCAAGGGTTGCAAAGGTATGATCCCTGACCGTGGGCTAA TCCAAGAGCAGCTAGCAAGCTACTCCATTCCCTAAAGTCAGAATATCCAGGGCAAACTTC ACACCCAGCCACTTAACGATGCCGTTGCCTGCTCTGTTTAATTAGAAGTCATGTGACCAT GAATGGGCCTGTATTTTTTCACCATTTTCAGTGATGAAATGGAAGGGATTAGTGGCTCAG CAATCCCCCTCCTGCCCCAGGAAAGATGTGAGCCAGCAGTTCTGCCATGAGCAAAGGCCA CTTTGGTCTGATTCTTTGCTCTAACCCAACAGGATAGCAACCCATCCCCACCATGAAGCA GAAGGCAAGGATCAGCCTCATCACAGCACTTCCCCCTCTGCTTGCTGTGCAGCTCCCGTG GGCTCCCTGGAAGGGATCAGACAGGTTCTTCTCTTCCTCCCTCATAGCCACTCATGCAGA GGCTTGCACATCCCATACATTCAGTATGTACCCTTGGTACAGAATTGAATTTAGTCAAAT AAAGGGTGACTTTAAATTTTCATTTGAGCTATGGTTAACTCAGAGCTGCCCATTCTGTCT TCCATTCATTTACAAATTTGCAGAGCATCTGTTGCAGCCAGGCACTATGCAGACACCAGG GTTAGAGCAGTGAGCAAGACAGACAAAGTTCCTACTCTTACAGTTTACAGTCCAGTGACA GAGGCAAACAGTTGGCCAAAAGTTGCACATATATGTTAATTCCTGGCTGGATAAATTGTC AAGGGAGAAAAAATGGGGTGAAGAGCAAAGGATGCTCAAGACTGAGCCTCGAAGAACTCT GATGTCTAAAAGTTGGGTAAAGGAGACTATAAAAGAGCATCACAAATGGCAAGAAAAGAA GCAGAAATGTAAGGCATGGTGGAGACCAAGAAAGCAACATTTCAAGGTAGAAGGGTACTA GGCAATATCAGACAAGCGTCCTTGGATCTAATATTATGGGAAGTCATTCATGATCTTAGT GAGAACAATCTCTGTCAGGATAGAGGCAAAAGCTCTGTTGATGTGGGCTGAGGAGTGAAT GAGGGACACTGATTGTACACAACTCTTTCAAGAAATTTTGCTTTGAAGGTGGGAAGAGAT CTAAGATGGTAGCTGGAGGGAGATGTAAGTTTCTGAAATATTCTGCTTTAAGTTAGGAGA AACTAGGTCATTAGAGATTTGTGCAATAAACCAAGAGACAAAGAGTAATGCACATGGACA TTAATGTTGCCTAGGATAATAGCCAAGAGTGGGCAAATATGGCCAAGAGCCTCACCCATC ATTACTGAGGCAGAGGGACCAAGAGTTTGGCAAGCGGCAGTAATGAAGAAGTGACAAAAC TAGGTGGCAAAGCTTCAAAGCAAGAAGGGGTTTTACAAGACACAGGAAGAGCAGTGGTGT GGAAGGGTCAGTGGTGAACAAGGAGAACGCCAGGCCTAACCCCTGAGGTACATGGGGTAG GGGAGAATGGCAGCCTTTGAATAGGGGGAGATATGTGCAGATCAAGGCAGGTGAGCTCCA TAGAGGTCAGGAGAAGTTTTGGGAGAAGGTCAAAGCGTGGGGAATGGGGTTGGGATCAAG GACTCACAGAGCAGTAAGATGTTCACAGCACTAAAAGAAGAGAAGATCACAGTAGGAGCT TTTTAAAGACACCATAAAAATAACCAGATATAAATGATCTCTTGGTTATCCAGGCCACTC TGCCTCCTTCCAAGATGCAAACAACCAAGACTTGACCCTACGCCCTATACCTGCATTTGC TTAGGCATTTGCACAGCACCAGGAACAGAGGGAACATTTCCAAGAATAAGTTTCCCCTTT GCACTTGACTTTACTCTCAGCAAAGTAGCTCAAATTAATATTTCAAGCAGCTCTTGAACT CTTGCAATGTGCAGGGCACTGCAGGGAAACAAAGTTAAGGAAGGTATAGCCCTTGTCCTC AAACAGCTCATTGTCTAGTAGAGAAGACGCATATACAAAATGCTAGAACTCGAGGCTGAC TGAAAAGTGGGATCCCCAAAGGGACAGGGTAGTGTGTTGTCACGTGACTAAGGGACACTC CATGCAGTAAGTGGAGCTTAAAAGAGATGGATTACTCAAGACTGTTGGAATTTAGACAGC CAAGAGAGGAGATAGGAGGGCATTGTGAATAGAGGCAAGGGCACAGACCAGGACAAAAGA AGGGCCTGGTGGCATCAAAGGGCCAGGAGAGGTATTAAGGACTTGAACTGGGGCAATGAG GTAGAGTCAACTAAAAGTGAAATTTTGGAAGAGGTAAGGATAGTATTTCGCTTCTGTTAA GATGTGGAGTGTGATGGAGACAAAACTCATCTACAGATAACACAGATGCCTGGTTTGTGC TGGCCCAAAGGCCCTGATGCCACTATCTCTTGTTTTCTCAGAACAAGTTCCAGGGTTTTG TCTTTGACATCGTGACCAGACAAGCTTTTGACATCACCATCATGGTCCTCATCTGCCTCA ACATGATCACCATGATGGTGGAGACTGATGACCAAAGTGAAGAAAAGACGAAAATTCTGG GCAAAATCAACCAGTTCTTTGTGGCCGTCTTCACAGGCGAATGTGTCATGAAGATGTTCG CTTTGAGGCAGTACTACTTCACAAATGGCTGGAATGTGTTTGACTTCATTGTGGTGGTTC TCTCCATTGCGAGTAAGTGGGCAGCCACGTGGTGGGGAAGCCCCACCTCTGTTTGGGGGT CTTTGGGATGCTCTTCTAAAACCAAGGAAATAACCAACCAACTTTTTGAAATACGTGTTT ATGTTAGAAGAACCCTGTATTTGCAGCTTTCCAAGAGTAGCAAGTTTTAAATGCCTTGGG CTGCTCTCTCAGTGAAATGAAGAGGTTCTCTTGGCCTTCTCCATGCTCACACGAAGTGTT TTCAGGCACCTACTGTGTGCACAGCCCATGGCTAGAACCTCAGGGGCTGACAGGGAATAA TCACTCCTCCTGAGAACCTCAGACCGTAAGATGATGGAGTCTCCAGCAGACCTTACTTTT ATGGTCTTGAAAGCAGGGGGCAGGTGGTGTAATGTGAGATGAGTCTTAAAATCTGTTCTG GAAAAGGAGATGGATTGCCATCAAAGTGAGGAAAGACTGGGAGGAGATTGTTCAAAGGAG ATTCTCAAGTCTAGGGTCTGGAATTAAATCTCTACACAACTGTTCTATTAGGGAACATGA GTGATAGAAAAGTCAAGATATCAATGCCAAGAAATTAATACTGTTAAGGCCCAGTCTGGG TGTTGTTGAATGACAGGCCCAGTATTTTGGGTCCAGTGGTGTTCAGAGAAAATTACTAGA GTTAGAGGCTAACATGGGGAAATGGAGCTGCCTTGACTGAATACTTCCTAAGTTCTGAGC ACTGAGCTGGGCTCCATGAATGTTCACAGCAGCCCTGTCAAGCAGGTGTTATTATCCTTA TTTTATACATTTAAAATTGAGACTCAGAGATCAAGAATATTATGCAGGGTCACAGAGTTT CCAAGGGTTGAAGCTGAGTTTAGAACAGGCTCCAATCTTCTAATGAACTTGCTGCCTGTC ACCAACAGCCAAGAGACTGTCCCTGGGCTTATGGGTTAGCTGAAGTTTGACCCTATAAAG GCCAAATCATGCTCACCAGCTTCACCTGGCATTATAAATGTATCTTGATTGGTTATATCC TGACCTGCTTACTTTTCATGTCGGGGTGTAGCTGTAGATTTAATTTGCCACTGATTTAGT AACAAGAGCTGAGAGTATCTAAATACAACTTAACAGCATTTCATCAACCTCCCCACCTTG GTGCTGAAACACAGCCTTGGGTTGCGTGTATTTCCGGGGATTTCAGTTTGTTTTTGTTCT TGTTTTATTTTGATATTGCTGCTCTGTTTTTTAACACTTTATTGAGGTATGATCCACATG TAAAAAGCTATACATGTTTAATGTACACAACTTTATGAGTTTGGAGATAAGTATACACCC ATGAAACCATCACCACCATCAATGCCAGAAACATGTCCATAACCTCCAAATATTTCCTCC TGCCTATTTATTTATTTATTGGGTGCTAGGAACACTTAAGATTTATTCTCTTGGCAAATT TTTAGGTAGACAACACGGTATTGCTAACTATAGGGACTATGCTATAAGTAGAGCTCTAGG ATTTATTCATGTCACATAACTGGAAGTTTGTACACTTTGACAAGTACCTCCCCAATTTCC CCTCCTCCCAGCTCCTGGAAACCACCATTCTATTCTCTGCTCCCATGAGTTTGACTATTT TAGATTCCTTATATAAATGCAATCATGTCGTTTTTGTCCTTCTGTGTCTGGCTTATCTCA CTTAGCATAATAGGAGTTTTTGTTGGTTGGTTGGTTAGTTGTTTTCATTTTTGTTTTGTT TTGTTTGTCTTCCAAAATGAAAGATTCCAAAGGCTATTTTGATTTTATATTAAAGAAGGG AGCTTGTTTGTGTATCAGTAATATAGAAATAAAACATAGCCAAGTACTTAGTTCTATGAT GGGGATATCTTAACTACCAGTTCCTGTGGCTTACAGGGTAGCACCATCCGCTGGGGCAGG AATCCCTTATAGAGCTGGAAAATAAGCAGAAAAAACCCACATATCAATCTTTCTACCCTC TCTCCTTACCTCCCTTTCTTCTTTCCCACCAACAAGAGGTGATTGAGTATCTAATTGTTA CAAGTACGAGGAACCATTCTCCCTTAAAATAAGATCCTGGAGCAAATTAAAATGTGCTGT AGACCAGTTGTCTGGCAGAAAATTTTACCAGAGTATGTAGACAAATAATGTACCCTACAC GATGGGAACATGTCCTAGTTAAACTGGAAATTCACAGAAGGTCTTCCACAAAGATTTTAT AATCCAAAATTATAATTATTTCCCAAGTCCCCTTCTACTTTCTTCCTCTTTCCAACACAA TATTGTGACCCATGTGCATAACAACAAAGAGCAAGTAGGGGAGAGGGCCCAGGGAACCTG GCAAGGAGCGAGAGCTTGCTGCTACATCCCTCACCATTTTTCCTGTGAGATGTTGAACCC TCTCTTACCTAAAGGCCTTGCCTCATCTTTGGTGGAGGCCTTTGGTACAAATAAGGCTCC CCAGGTCCCCAAGACCAAAAATCAAACATCTTCAAGTTAGACATCATATAGAGGGACATA TACATTCTTTATATTTCCAACTTTGTTCATAGTTGGAGTTGGGGGCAGTAACTGACTCCC CCAAAAAAACCTATCTCCAGGCCCTAACATAAAGTTCAAAAACAGCTGGAACCGAAAAAA AAAAAAAAAAAACAAATTAAGGCATTGTGCAGACAGCTGGTCATTCAAATCCCTTATGCT TTAGCTGTCAGCCAGTTGCCCGAATCCTCAGGCCCCCTTGTAATTAACACCAGGCTCCGT ACAATTAGCCACAGGACCGGGTGCCAGAAACAGAGTCAATCTTCACAGTAGGAGGTAGGG AGAGGAATCATAAGGGGTCCTGGCCTTCACAGAGCACAGGACCAGGGTGGAGGAAAGGGC ATGACCCAAAGGCTGCAGTATCAGGGAGGAGCAGGGTCTGTGTCATAGGAAAAAACAGGC CAAAGACTGTGAGCCTGTGGAATAAACTGCAGTCACTTCCAGCTGAACTTGGCTGCAGAA GATAAGTTTTTGACTGTGGAGGTTGACCAAGAGGATATTCTAGGCGGAGGCAAAGATAAC ATGGTAGGAAGGGTAAGACGGAGGAGGAAGAGAGGACTTATGCAATGGGCATTTACTGAG CCCCTACTTTGTGCTACCTACCATGCCAGGCTCTGAGGGCCCAGAGTCCTCAAATGTGGA TGCCCAAGTGGCAGTCTATACCAAGCAAAGCAGAGTGTCATCACAGCCAACTTTGTCACG TCTCATGGCTGGAGATACTAATTAGATAATTTCTTCCTTCTTTTAAAGGCCTGATTTTTT CTGCAATTCTTAAGTCACTTCAAAGTTACTTCTCCCCAACGCTCTTCAGAGTCATCCGCC TGGCCCGAATTGGCCGCATCCTCAGACTGATCCGAGCGGCCAAGGGGATCCGCACACTGC TCTTTGCCCTCATGATGTCCCTGCCTGCCCTCTTCAACATCGGGCTGTTGCTATTCCTTG TCATGTTCATCTACTCTATCTTCGGTATGTCCAGCTTTCCCCATGTGAGGTGGGAGGCTG GCATCGACGACATGTTCAACTTCCAGACCTTCGCCAACAGCATGCTGTGCCTCTTCCAGA TTACCACGTCGGCCGGCTGGGATGGCCTCCTCAGCCCCATCCTCAACACAGGGCCCCCCT ACTGTGACCCCAATCTGCCCAACAGCAATGGCACCAGAGGGGACTGTGGGAGCCCAGCCG TAGGCATCATCTTCTTCACCACCTACATCATCATCTCCTTCCTCATCATGGTCAACATGT ACATTGCAGTGATTCTGGAGAACTTCAATGTGGCCACGGAGGAGAGCACTGAGCCCCTGA GTGAGGACGACTTTGACATGTTCTATGAGACCTGGGAGAAGTTTGACCCAGAGGCCACTC AGTTTATTACCTTTTCTGCTCTCTCGGACTTTGCAGACACTCTCTCTGGTCCCCTGAGAA TCCCAAAACCCAATCGAAATATACTGATCCAGATGGACCTGCCTTTGGTCCCTGGAGATA AGATCCACTGCTTGGACATCCTTTTTGCTTTCACCAAGAATGTCCTAGGAGAATCCGGGG AGTTGGATTCTCTGAAGGCAAATATGGAGGAGAAGTTTATGGCAACTAATCTTTCAAAAT CATCCTATGAACCAATAGCAACCACTCTCCGATGGAAGCAAGAAGACATTTCAGCCACTG TCATTCAAAAGGCCTATCGGAGCTATGTGCTGCACCGCTCCATGGCACTCTCTAACACCC CATGTGTGCCCAGAGCTGAGGAGGAGGCTGCATCACTCCCAGATGAAGGTTTTGTTGCAT TCACAGCAAATGAAAATTGTGTACTCCCAGACAAATCTGAAACTGCTTCTGCCACATCAT TCCCACCGTCCTATGAGAGTGTCACTAGAGGCCTTAGTGATAGAGTCAACATGAGGACAT CTAGCTCAATACAAAATGAAGATGAAGCCACCAGTATGGAGCTGATTGCCCCTGGGCCCT AGTGAGAACACTCCAGCCTGGATATGTTCAGTTATGATGCGTCCTTCTGCTCTGTGTTAA CTCTTTCCCATTGCAGATCACACCCAGCTACAGCCTCACCAATGCATGCCACTGGTCACA ATGTCAGAACTGGGCAGGGAATGCAACTGGTAACCACCTTTGAAAAAACCATCATACACA AATAGGAGTCAGAAGCTAAGAGCACTTCCACTGTGATTTCCCTTCTGAATTTCAATATCA GATCATGGGTTCTCTTACTCTCCAGAAAATATCCCTTGTCCTTTTATTTTTGTTGTAATC AGATTATATTTACTGAGACAAACTTCTCAGGACCAGAACTTCCAAAGATATAGAACAAGA ACATTACTGAAGGTCCAAGGTAGTCCTCTGTGGAGCACCATACAGAGATTGGCAATATTA TTTAGGATTTTGCATGACTGCATGTGAGAGCTGTCGGACAACTGCTCTGACCCAGGGTAA CACCTGTGGCCTATGTCATGAAAAGCCTTTGAGATATCCATTAAAATTAATATTTTTAAA TGTATGTCCACACCAGCGTTATATCATAAAGCATCATTTTATTTTTCTCTTATTTCTGTG ATGCTGACCTGTATCATGAGTCACTTACCCCTCACTTTCTGGGATCCTGATATTTTTCAG TTTGAGATTGAGGCTGGGTGGAGCAGGAAAGAGCACTGTCCTGGGATCTGGAAATGCCAG TTGGAATCCTAACTTTTCCACCAATTGTCTGGCTGACCAGGGCTAAAGTCTTTCCCCTCT TAACTTCAATTTCCTCACTTGTAAAATTGGTGGGAAGGGAAGAAAATGAATGCTTTCTAT AGACTTCGCATCTCGTGCTGTAGAACTCCATGGTGCCTGCCCCATTTATATGGGAAAAGC ACTCTTTTCTGAGGCTGAAATGGCTGGACCTTTCTGTTGTGTAGCTAAAGAGGCAGCTTT TGAATACATTCCAGAGTCTGTTCACATCTCATAATCAGACATTAAGGAGGAGAGTTTTTA CATTACCTGGCGTCCAGAACTCCATAGTTAATATAATCAGAAGAAAAAGTATAATCATGA GAATGATGATCCAGTAGATAAAAGGTATGGCCAGTAGTTTCTTTCATTCATCCTGCAAAT GTTATTAATTACGTACTATATGATGGGCACTGCTCCAAGAACAAGAGATAAAATAGGAAT TAAGCCCCCAAAAGATTAATTGTATGAACTGAATGGAGTAAGAATTGTCAGAGGAGTTTA TTTAGGCCAAAGGCATGTTGCAGATTTTGAGATGAAGAAAAGCAAAGACTCCCTATAACA ACATAGGACATAGGGATTGTAATGACAGTCATGATCTTCCATTAACTCTTGGCCCATTCC CAGCCACAATCACAAAGATTCAAATATATAGCCAATTCATTGTCTATATATTTGATCATT TCATAAGATTTTCCATTTGTATAATTACAAAGCACAGTGGCTGATACATCTCACATGCCT CAAGAATACAGATTTGCCGAGGCCTTGCTCTCCCTTTTCTGTCACTATTAGCCCATAAGA AGTCAAAGAGGCTCTCAAGATGCTTACTGGGAGTTGGCAGCATCCACAGATGCAGGTTAA TAAGATGATGATGGGATAGCAGAGGTACCAAGCCATCTCAAATCAGCTATTCTCTGAATG CCTGATTTATTCTAAAAGATGAGTTCAACCAAATAACTCTTCCTCTCTCGTACCACTGAA CCCGGGAGC 137 Human Nav1.9 MDDRCYPVIFPDERNFRPFTSDSLAAIEKRIAIQKEKKKSKDQTGEVPQPRPQLDLKASR amino acid KLPKLYGDIPRELIGKPLEDLDPFYRNHKTFMVLNRKRTIYRFSAKHALFIFGPFNSIRS sequence LAIRVSVHSLFSMFIIGTVIINCVFMATGPAKNSNSNNTDIAECVFTGIYIFEALIKILA RGFILDEFSFLRDPWNWLDSIVIGIAIVSYIPGITIKLLPLRTFRVFRALKAISVVSRLK VIVGALLRSVKKLVNVIILTFFCLSIFALVGQQLFMGSLNLKCISRDCKNISNPEAYDHC FEKKENSPEFKMCGIWMGNSACSIQYECKHTKINPDYNYTNFDNFGWSFLAMFRLMTQDS WEKLYQQTLRTTGLYSVFFFIVVIFLGSFYLINLTLAVVTMAYEEQNKNVAAEIEAKEKM FQEAQQLLKEEKEALVAMGIDRSSLTSLETSYFTPKKRKLFGNKKRKSFFLRESGKDQPP GSDSDEDCQKKPQLLEQTKRLSQNLSLDHFDEHGDPLQRQRALSAVSILTITMKEQEKSQ EPCLPCGENLASKYLVWNCCPQWLCVKKVLRTVMTDPFTELAITICIIINTVFLAMEHHK MEASFEKMLNIGNLVFTSIFIAEMCLKIIALDPYHYFRRGWNIFDSIVALLSFADVMNCV LQKRSWPFLRSFRVLRVFKLAKSWPTLNTLIKIIGNSVGALGSLTVVLVIVIFIFSVVGM QLFGRSFNSQKSPKLCNPTGPTVSCLRHWHMGDFWHSFLVVFRILCGEWIENMWECMQEA NASSSLCVIVFILITVIGKLVVLNLFIALLLNSFSNEERNGNLEGEARKTKVQLALDRFR RAFCFVRHTLEHFCHKWCRKQNLPQQKEVAGGCAAQSKDIIPLVMEMKRGSETQEELGIL TSVPKTLGVRHDWTWLAPLAEEEDDVEFSGEDNAQRITQPEPEQQAYELHQENKKPTSQR VQSVEIDMFSEDEPHLTIQDPRKKSDVTSILSECSTIDLQDGFGWLPEMVPKKQPERCLP KGFGCCFPCCSVDKRKPPWVIWWNLRKTCYQIVKHSWFESFIIFVILLSSGALIFEDVHL ENQPKIQELLNCTDIIFTHIFILEMVLKWVAFGFGKYFTSAWCCLDFIIVIVSVTTLINL MELKSFRTLRALRPLRALSQFEGMKVVVNALIGAIPAILNVLLVCLIFWLVFCILGVYFF SGKFGKCINGTDSVINYTIITNKSQCESGNFSWINQKVNFDNVGNAYLALLQVATFKGWM DIIYAAVDSTEKEQQPEFESNSLGYIYFVVFIIFGSFFTLNLFIGVIIDNFNQQQKKLGG QDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNKCQGLVFDIVTSQIFDIIIISLIILNM ISMMAESYNQPKAMKSILDHLNWVFVVIFTLECLIKIFALRQYYFTNGWNLFDCVVVLLS IVSTMISTLENQEHIPFPPTLFRIVRLARIGRILRLVRAARGIRTLLFALMMSLPSLFNI GLLLFLIMFIYAILGMNWFSKVNPESGIDDIFNFKTFASSMLCLFQISTSAGWDSLLSPM LRSKESCNSSSENCHLPGIATSYFVSYIIISFLIVVNMYIAVILENFNTATEESEDPLGE DDFDIFYEVWEKFDPEATQFIKYSALSDFADALPEPLRVAKPNKYQFLVMDLPMVSEDRL HCMDILFAFTARVLGGSDGLDSMKAMMEEKFMEANPLKKLYEPIVTTTKRKEEERGAAII QKAFRKYMMKVTKGDQGDQNDLENGPHSPLQTLCNGDLSSFGVAKGKVHCD 138 Human Nav1.9 AAGGTTCTCCAAGGCCCCACCAGAGCAGCTAGATACAGAGTGTCGATTGGTGCACTCACA nucleotide AACCCTGAGCTAGACACAGGGTGCTGATTGGTGTATTTACAATCCCTGAGCTAGATATAA sequence AGACTCTCCACGTCCCCACCAGACTCAGGAGCTCAGCTGGCTTCACCCAGTGGATCCCGC ACCGGGGCCGCAGGTGGAGCTGCCTGCCAGTCCTGCGCCATGCGCTCGCACTCCTCAGCC CTTGGGCGGTCGATGGGACTGGGCGCCGTGGAGCAGAGGGCGGTGCTTGTCGGGGAGGCT CCGGCCGCACAGGAGCCCATGGAGGGAGTGGGAGGCTCAGGCATGGCGGGCTGCAGGTCC CAAGCCCTGCCCCGCGGGAAGTCAGCTAAGGCCCGGTGAGAAATCGAGTGCAGCGCCAGT GGGCCGACTCTGCTGGGGGACCCAGTACACCCTCCGCAGCCACTGGCCCGGGTGCCAAGC CCCTCACTGCCCGGGCCGGCAGGGCCAGCCGGCTGCTCCGAGTGCGGCGCCCGCCAAGCC CACGCCCACCCGGAACTCCAGCTGGCCCGCAAGCGCCGGGTGCAGCCCCGGTTCCCGCTC GCGCCTCTCCCTCCACACCTCCCTGCAAGCTGAGGGAGCCGGCTCTGGCCTTGGCCAGCC CAGAAAGGGGCTTCCACAGTGCAGCGGTGGGCTGAAGGGCTCCTCAAGTGCCGCCAAATT GGGAGCCCAGGCAGAGGAGGCGCCTAGAGCGAGCGAGGGCTGTGAGGACTGCCAGCACGC TGTCACCTCTCACTTTCTTAGGGTCGAAGTCCTAGAAGTGAGAGAGCCGGCATGGGGGTC GCAGTTCATGCCTATGTTTATGGCTCTTTTGTTCCCATTTCATCACATTTTAAGAACTTT ACTGACTGGATGTGAGAAAAGACCCATGCACTTTCAGTTTTGCTTTTGCAATCCATAACT ATGGCCCAGTCAATTCCCTGTTCCTTTTTCTCTTGTTTGGGCCATGTGGCCACTTGCCTG CTACTTGCAGATCCCACTTCAGCAGTTCAGCCGGCTCAGCCTTATTGTCTTGCTTAATGT CTGGGTCTCAGTTTTAGAGACTGGGCTTCTTCTGCTCACTTGTTCCTGAACTCAACTTCC CGCCTTTTGCCAGGTCCTCCAGGAATGATGCCCTGCTTTGGTTTTGTCTATGCCAAAAGT TCCTGCTATACCCACAGTGGTGGTCATCTCTTCTGATCTTCACAGCCAATCAGCTCCCAA GGCCCCTGACCTCAGCTCAGCTTTTGTAGATCCTTATGACACCATCCTTTAAGACTGGAA TCCTAGGGCAGGCTGTTTTATTCCCGCCTCCTGAGGCCTTTCTGAGGATCTGTGGCTTGT CTCTGTCCTGAGGGTGAAGATGGATGACAGATGCTACCCAGTAATCTTTCCAGATGAGCG GAATTTCCGCCCCTTCACTTCCGACTCTCTGGCTGCAATTGAGAAGCGGATTGCCATCCA AAAGGAGAAAAAGAAGTCTAAAGACCAGACAGGAGAAGTACCCCAGCCTCGGCCTCAGCT TGACCTAAAGGCCTCCAGGAAGTTGCCCAAGCTCTATGGCGACATTCCTCGTGAGCTCAT AGGAAAGCCTCTGGAAGACTTGGACCCATTCTACCGAAATCATAAGGTACTTCATTGGGC GGGGGGGGGGGGTGGGGGTGGGGGTGTTCTAACCATGCAGTTGTAACTGGTGTTACAGGT TCTCCAAACACCACTCTTAGGGTTTATAGCAGGCAGGCTGTGCCTCTTCTTTGCTGTCAT CCCCTGGCATTGCCTTGATAGTCCTGAAAACCCCTTTCAGTCCTCATTTTTTCTCTGCAA TGCATGTATTAGAGGAGAAGATGAGGATGAGGGACTGCAAAAGGATAAAAGGAAAACAGA GAAAACACAAAATGAAGTCTGGAAAGAGGACTGGACTAGTAGTCCCAGAAGTTAGATACA GGCCATTTCAATTGATTGGCTTTATGACTCTGGGCAAGTCACTTTACTAATCTTCACTTT TAAAATCTGTATAATAGAAATAATGCCTGATGTTCCTACCTTAGAGCATAGACATGCTGG GAAAGCACTTTATCACTATAATGCATTATACTGATACAAAGGATTAATATCATGGTCCAG AAATGCTCATTATAGCAACAAGATTTTACCTTAAAAACTCTTGGATAATAGGCTTTCTAG GCTATTAAATGAGACTAATAGGGCTCAAACCAACAACTGATGCATAGTAGGAGCTCAATA AATGAAACCTAAGAAAATGGTAACCAAGTGGGCACATGCAGATTCTATGTAAACTCAATG CAAATATGTGAAAGCCTTGCTATTTTATCTGCCTGGATGCAGTTCTTGCAGGGAAATTCT AGTAAAGGTTTCTTGAAGGGGCTCCCGAGTGAGGGAATGTAAACTTTGAGCTCCCACATG ACTGCTCTAGAGCCTGAGGCTGTGAGCACACTTGGCCCTTTCAGGAAAGGCTCAGAAGAT TTTGCCATCATCACCTCGCAGTGTGATCCAGCAGAGGTTGGAAACCCCACCTTGCCATAC TGAGGTTAGAGACCTAGGAGGCTGGGAGGTTTGGGGAAGCCCTAGAAAATATGGGATGCT GAGAGCATCTTTGGCTGGCATCCATAAGAAACTGACCTTTCAAAGAAAGATTGCCTGATT TGCATGTACTTCAATTGGGATAGACTTAGAGTTGCCAGGCCAAATGAGGACCACATATAC AGAGGAAAGTAGAGTATAAGTAGGAAGGACAAGCAAGGGGGAAAGAATTGAAGGAGCAAA AACACTCCAATGTTTCTCCTTTTTCTGATGCCCCAGGATTCCAGGGCCTTCTTGGAGGTA TAGTTGTTGGAGGTGAGTTAACTGTAGAGGTCTCCTTCTGCATCAATGCTGGAGAGGAGA CGCATGAGTCAGGCAGCAGTTTGGTGTGCAGCTGACAGTCCGGGAGAACAGGACTATTCC AGCCTGGGTGGTAATTCTAGGACCACCACTTCTCAGCCTGGGTCCTCACTTTAAATAAGA AGACACGATATCTGTTTAGAGCTCTGCACACCCATGATCTCACATGACCAGTGCCCTCAA CAGTCATGTGAGGTATGTGTGATCATGATTAGCACTCTACCTTACAGATGAGGAAACCAG AGCCTAGCTGGGAAAGTGGCTTAAGCCAGGTCAAGCAGCCAGTGAGTGGTGGAGCTAGGA TGCAAACCCAAGGGTGCTTTTTATCATCACAGGCTACTTCTCTTTGGAAGCCCCATTTTG AAGAGCAACAGGGGAAAGAAGTAGAGAAGTGTCAGCAAGCAGGCAGGAGGAGCTAGTACC TATCATTTTAATCATTAAAACCCTGGCATGTAGAGTTAAATACACCATTAGTTAAAATAT TTAAAGCAGTAAGAAGAGGCATCAATAAAATATCAAAATCAAAGGCACAGAAGGAGAGCC ATATTGTACCACTTATTAATGTGGGGTGACTCATCTCAAAGAGAGGTTTGTTTCTGCCCC AAAATTGTATGTCTGTTCAAAGGAAAGGCGCAGAATTTCCAAAGCAGGTCTTGGGCTCCT GGCTCATATTTTTAAAAAACCCCGTGGCTCATTTCATTATTATGGGGGCAAAGAAAAATA CCCTAGCACAGTTTTCCCTGCAGCCAGATAACCATCAGACCAGTCAATCCCAGCAGCTCA TTAAAGGAAGGTTGATGGTTGGCAGTGGCTGACGTTCTGGCACCTGAGCCAAGGTAGCAC TCACAGTGCTAACGTTGGGCCTGGGTGCCGAGGTCTGTCTCTCCTGCCTGAGAACATGCT GTATTACATGGCCAAGTAGTACTTTTTGCAAATCTGAAAGGAGCCATCAGTTGGGGAGAA AGGAATGACTCTTACTTTCTGAAGGCTTTTGTAACTGATAGCCATGGGGACAGATATGGG GACAGCTAGATTACAGTTACATGGAAAAAGGGAGGTCTGGAGGTGGATGTTAGAAGCATG TTAGTGACAGGTTGTTGTCCAGGGGCTGCTTCATTCCCAGTGCCCAGGGAGTCATGCTAG GAGAAAATGGGCAGAGGTTCAGTGTTCTTTAATGGGACTCCACTGGGCACTTTGTGGGGG GGCGGGCATAATGTACCATTGGCAAGTTTGTCCTGTTTGGCATCCCTGGGTCTTCCCAGT AAAAGGCCGGTAGAGCCCATAGTCATTACAGCAGACAACCAAAAATACTCACACACATTT CCAAATGCCTTCTGTAGGGGCAATGCCCACCTCCCCTGCTGGTTAAGAACCACAGGGAAA TTTGCCCCTATGTCCACAGAGGTAAAGAAAGAGCAAATCCAGGAAACTTGGTGACTTTTT TCTTGAAGATCATCAAGGTTAGAATTGACTGGTTATTTCACACAGCATTTCTCAGACCTG TGGCATAGCAAACATGGACCCTTTAGGTCACTTATTCTAAGATTCATTGCAAAGTTTTCG TTAATAATCAAAATAATTTAAAATAATCTGCTGAATCTAAATGTCCAAGCTCAATTTCTG CCTCTTCAAGTTCTCTTCTAGTCCCTTTCCCCGTTGATACGTACCATGGAGTACAATATT AAGGGTTTTCTTTTCCCCAGTTATTCCTCTATACTCCTAATTTTGGTATTTTCAGGTCCT GTGTTTTCCATCTCTGATTCTTCTGGAGTATCCCATTGGGTAAATATTCCACAGCCCAAC AGTTTATGTTATAATTTTCTCTAAATGAATTCATGATAAATATTGTTTTGTCACTGCATT TAATTCCTGTTGTGTATGTGTGTGTTTTCTTGTATTGTTTTTTTTGTTTTGCAGACATTT ATGGTGTTAAACAGAAAGAGGACAATCTACCGCTTCAGTGCCAAGCATGCCTTGTTCATT TTTGGGCCTTTCAATTCAATCAGAAGTTTAGCCATTAGAGTCTCAGTCCATTCATATCCT TTCATTGCACCTTTTACTAAGTCCAGATCATTTGAAAAGTGCACGTGCCCCACGGTGTTC ATGTTATTGGATGGAAGCAAATAGCTAATTCTTTCTACAAATACTGGCTGAGTGCCTACT GCTGCAGGCAGGGACTGTGCTGGATGTGAGCAAGATGTCGTCTCCTTCTCTGGGGTCCTA TGGCCTCAGAAGGGCATAGACTAAAAGGCAGGCAACTTCTTTAGTGAGATTGGTCGTGTG AAAGTGGTAGCAGGGAGAGGCACTGCTGTGCTTTAGAGGCATCAAGGATGGCTTCTTAGA GGAAGGTGCATCTAAGCTGACACCTAAAGAGTTGTAAACTGGCAACCTGTGCAGTATATT GAGTTGATAATGTGTTGTGTTGGACCTACACAGTATTTAAAACTTTGAAAAACACTTTCT AACATGAATACTCCAGATTTCCAGCTTTTCTTGAAAGATGGAAACTGGCAAGGGTAGGTT CCCATTTCTGTTAATACCTGACAAAAATGGGTAGAGTAAGGAATTGCTGCCTCTTTGGAG GGGGCATGTAAGCTCTGTCTCACCACCTCTGCACCCCTCTTGTGGCTGAACACCAGCCCA TTTTACCCATTTATATGCCTTTTCAGGCTCTTAAAACTTAGGGTTTGCAATGTCAGTCTT AAAATGTAAAGGCAGGTAGGAATTATTAAGCATTTCTGCTGTATGCCAGGGTTGGGGGAG ACACTAGGAAGTTTTAGACAGGGAAATGAGAAAAAGATATGGGTTGGTAGATTTAGTAAA ATATTGAGGAGAATGGCAATGAATAACTATTTGGGTTTGGTTCCCTAGAAGCAGAGCCTG ATATGGGCATTCTTATTTAAGTGATTTAACAGAGGAGTGCTTTCTGGAGAAGGAGAAAGG GAAGCAGAAGGGGGGAAAACAAAGAGCCAAGCAAGGATATGGTCTCAGCTGAGTCACCTT CATTCTGATCCCACGAAGAACTCTGGAGCATGAATTGCACCACAGAATTGTCCCACTTTG AAGCAAGGGCCTAAGTCAGTCAGACATGTAGAAGGGGGTGTGTAGCTCTCCAAGTGAGGA GGCTGCCATTCAGCAGGGGACAATTCTCCACAGGAGGGGACAGTTGTGAGTTGTTCTCAT CCAACATTCACAGTCAGAAGATAGATGCACCTGCAGAAAAGGGGACCATCAGGGGCACCC CCAGTCTTACCCAGTAGCTAATGATGCATGCCATCCTGCAACATATCTGCCTTAATGGAT GCTGGCCACATTGTTCAGCATGTTCATTATCGGCACCGTTATCATCAACTGCGTGTTCAT GGCTACAGGGCCTGCTAAAAACAGCAACAGTAACAATACTGACATTGCAGAGTAAGTATT TTTCCTTCACTCACCTGTCCTAAGTTTTTAAACAAATATACCTAAGACAGTTAATGATTA CACTGGAGAGAAAGAAAAAGTCACTAATAATTCTAACACATATGTTGGCAGAATCAGTTT TATTTTTGACCTTTATTTTTTTACAATTTGTAATGAACAGCACACATATGTTCACATTTT AAGTGGTTTTCTTTGTGTTTACATAAGTTTTATAGGTATCCATTTTGATGGTTATATAAT ATTTTATCAGGTGAATAAACCATAATGTACTTCACCACTCTAGCATTTTAGTAGATTGTA ACATTGTCATAAAAATCTTTTTGCATAAAGCTGTATTTATGTATTTACTTATTCTTTGTT TTTGTTTGTAAAAAATTGTTATGGGAGTTCACTATGCCAAAAATAAAGCTATTCTTTTTT TATTTTTTTTTATTTTTTGAGACAGGGTTTTGCTCTTGTTGCCCAGGCTGGAGTGCAATG GCACTATCTCAGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCGATTCTCTTCCCTCAG CCTCTCAAGTAGCTGGGATTACAGACGCCTGCCACCGTGCCCAGCCAATTTTTTTGTATT TTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTTGAACTCTTGACCTCAGG TGATCTGCCCACCTTGGCCTCCCAAAGTGTTGGGATTACAGGCGTGAGCCACTGCACGCA GCCCTATTCTTAAATTATTATTTTCTTGAGATACACTCTTAGAAATGGATTTTCTAGGCT GGGCGTGGTGGCTCATGCCTGTAATGCCAGCACTTTGGGAGGCCAAGGCGGGTGGATCAC GAGGTCAGGAGATCAAGACCATCCTGGCTAACATGGTGAAACCCCATCTCTACTAAAAAT ACAAAAAATTAGCCAGGCGTGGTGGCGGGTGCCTGTAGTCCCAGCTACTCGGGAGGCTGA GGCAGGAGAATGGCATGAACCTGGGAGGCGGAGCTTGCAGTGAGCCCAGTGAGCCACTGC ATTCCAGCCTGGGCAACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAATGGATTTTCTAG ATAAAAAGGTACATTTTAATGCTTTTAATGATATATATAGACAAAATGTTTTCCAAAAAG TTTGTATCAATTTGCTCTGCCATCAGCAGTGTATTCTAATAACCAATTATATCACATTCT TGCTAGCATTAAATTTTTTCTTCTCTAATTTATGGAGTTAAAAATATTAGCATTTCTTTA ATTTCCAGATGGGTTAGACTTAAAACAATTTTAATTGACACATAATAATTGTACACATTT CAGGGTACATGTGATATTTTGATACATGCATACACTGTGTAATGATCACTTAAGGGTAAT TGGGATATCAATCCCAATTGGACATTTATTATTTCCTTGTATTGGAAACATTTCAAATCT TCTCTTCTAGCTATTTTGAAATATACAATAAATTATTATTAACTAAATAGTCATCCCACT GTGCTATTGAACACTAGAATTTATTCTTTCTGTCTGTTTTTTGTACTCATTAATCAACTT CTCTCCATTCCTCACCTCTCACCCAGTCTTTCCCCATCTCTGGTAACCACCATTCCATTC TCTACCTCCATGTGATCCACTTTTTCAGTTCCCATATATGAATGACAACATGAAATATTT GATTTAGACTCTTTTAATGTTTTCATTTCTGAATTGTGTGTTTCCATCTTTGCTTCTGTA TCATTTTTATTTCTTTTCTAATCAGTTTGAGTTCTCTGTATAACCAAAATATTAACTTTT TATCATTTGCATTGGCTTTTCAGACATATGAATTAATGTTTTAACTTAAAAGTTCTATAT TTGTATAAATATTTTCTTATCAAAAGTTCCAGTTTTTCTAGTTTAACATAATTACTGCTT TAATCTTTCAGGAACATATTGAATGCAAATTGAATAAATGAGACCATTTTTTCTAACTCC TATTTAATTATGCAATATAAACCACTGCAATCGATATATAGACCATTTCAATTACCCCCC AAAACGTCTCATGCCTCTTTGCAGTTAATCCCCGATTCCCACTCCCAACCCCAGACAAAC ATCGATCTCATTTCTGTCATTCTAGACTTGTTTTGCCTTTTTTAAGAATTTCGTATACAT TTCATATGCTATGTATGATACTATATTTTTGTATCTGGTTTCTTTCATTTAGCATAATGC TTTGAGATGCATCCATGTTGTTGTGTATATTTCTACATTTTTTTTTTCTTTTTGTTGCTA AGTTGGATTCCATTGAATGGATATAGTTTTCCCATTTATCTATTGATGCACATTTGCATT ATTTCCAGTTTAGGGCTATTATGAATAAAGTTGCTGCGAACACTTCCGTATAAAAATCTT TTGTGGATATTTTTGCATTTCTCTGGGATAAATACCTAGGTGTAATTGCTGGGCCCTGCG ATAAGTATATATGTAACTTTCTAATAAACTGCTAAACCTTTTTCAAATTGACAGCATAAT TTTACATTCATATCAGCAATATATGAAAGTTTCAAAGTCTTTATTAATTTTTTCATTCAA TTTGCTTTGATTTTAATTTGCTCTTCTTTTTCTAGCTTCTTAAGTTGGAAGCTTAAATAG TTGGTTTTAGATTTTTCTTCTTATACAATTGTATAAAGCTATGCATTTCTGTCCAAGAAC TGCTTTAGCTGCATTCCACAGATGTCAATACACTGTGTTTTTATTACCTTTTGGTTCAAA ATATTCCCTACATTTTCTCATGATTTCTTCTTTGTCCCATATGTCATTTAGAAGTGTGTT TTTTAATATTTCCAAATATTTTATACCTTTCTAGTTGTTTTCTGGTTGTCTGATTATTAT CTAATTTCAGTTCAGTTATGATCAGAGAATACACTCTGTAAAATTTCAATCTTTTGAAAT AAAGAGAAACATCTTTTATAGTCTATCTTGGGAAATGTTTCACATGCACTTGGAAAGAAT GTGGATTCTGCCACATTCTTAAATACTCCTTAAAAGGTCAGTTAAGTTAAGGTGGTTGAT AGTGTCATTTAGATCTTTTGTACCTTTACTGATTTTTACTTAGCATTTTTATGAATTACT GAAAGAGAGGCTTGAAATCTCTATCTATGATTGTGGATTTGTCTGTTCTTTCTCTCTTTT TGTCAAATTTGGCTTTGTGTATTTTGAAGCACTATTATCAGGTACATACATATTTACGAT GATTATTATTATCCTCCTGATGTATTCATTCTTTTATTATTATGGAATGTCTTCTTTGTC TCTAATAATACTCATTTAAATATTTGTTTTGTCTAATATTAATATAGCTACTTCAGCTTT CTTAAGCTCTTTGTTTGCATGGTATATCTTTTTCCATTCTTTTACTCTCAAACCTTCTGT GTCTTTGTATTTGTAGCGTATATCTTGTAAACAACATATAGTTGGTTTTTGCTTTTTATG CAGTCTATCAATCCAGTCTCTGACTTTTGATGACTAAAGTTCATTTACATTTAATATAGT TATATAATTGGATTTTGCTTTGCTATTTTTCTATTGGTTTTCTATTTATCTCTTTTTTTT TTTGTCATTGTTGTTGTTCCTTTGCTACTCCATTATTTCTCCATTCCTGCTTCCTTTTGT GTTAATCATGTATATTTAGTATTTCATTTTAATCCTCTGCTGGCCTTTTAGCTTTACCTA TTTGCATTTTTTAGTGGTTTCTTCACATATTAAACAATGCATTGTTAAATTGGCAAAATA GAGTTAACGTTGAATTCCTTCAAGTGAAACAGGAACCATGCCACAGTTTAGTCCCCTGTT TGTTTTTTATCATATAGTTATGCTAATGTACACAATAAACCCAACAATACAGTTTATCAT TTTTGCTTTTAATGGGCACATGCCTTTTAAAGACATTTTTTTTCCTGTAGATCCAAGTTA TCTTTTGGTAGTATTTCTTTTTAGCCTGAACAACTTCCTTTAATATATCTTGTGGTTCAG TTCTGTTGGCAACCCATTCTCAGTTTATATTGATCTGAAAATGTCTTTATTTCATACTCA TTTATGAAGTAGAGTTTCATTGTATATAGAATCAGTGCTTGACAATTTGTTTTTCTAGCA CTTTAAGTATGTCATTTCCATGTTTTATGCCTCCTGTTGCTTAGCTCTCTCTTGCATGCT TCCAGGATTTCCTCCCTAAATTTCCAGCTGCTCTGCCAGCTCTGAACCCCACCCTCGGCC ACCTTAAATCTTCAGGACTGCAGCCATTTGCCAGTGTTGGGGTGGGAGTGGGGAAGTTGG AAAACACTCTCAGACCAAAACAGTTAAACATTTTCATAATTCTTATGCATTGCAGGTACC TTCTTTTAAGGGGAAACTCTGCCCCAGTTTCTGCCTCCTTTTGTTTACCTTCCAGGGCCT TCAAATAATTGTCCTTATATATATATTTTCCAGTTTAATACTCATTATCTGTGGGAGGGT TTGCCCAGCTACTTCATGTCTTCATCACAATTGAAGCAGGCATCAATGGTCTCTTAATTT TGCTTATTATTTTTTTCTTTTCCTTTATTAAATTTAACAAACTTTCCTCTATGCCCGTGG ATTTTGATTTATAGTTAGAAACACTTTCTCTAGCCTTGAGTTATAAAAGGACTCTCTCAT ATTTTCTTACAGTGCTTTTATAGTTTCCTTTTTTAAAAATTTAAATCCTTGAATAATTTG GAATTCATTCAAGGGTAGGGTGTCATAAATAGATCTGACTTCATAGTTCCGTCCCAGATG TCTATCCATTTGTCCCAACACCATTTGTTAAATAGTCCACCTCTTATCCTCTGATGTGAA ATGTTACTTAGGTATTCAGATCTATTTCTGGACTTTCTTTTCTCTTCCATTGGTCTGCGT TTTCATATGCCAGAACCACACTTTTTAACTATCAAGATTGTCTAATGTTGTAATGTCTAG GAGTGCTAGAGGTTCCTTTTGAGAATTTTTATGGCTCTTGTTGATTTTTTCATGTAAATT TTATAGTCAACTTGGTTAGTTTTATTGACATGATGAAAGGATTTATAAATTAACCTAGAG GAAACTGACATCTTTATGAGGTTGGAACTCATTGAGAACATGACATATTTTTCATTTGTT AAAATCTTCTTTTGTGTTCCTCTAATTGCTTTAAAGTTTTCTTCATACAGATCTTCCACA ATTCTTGTTAAATTTGTTTGTGAATTTTACTGTTTTTTGTTGCCATTATAAATGTGGTCT TTTCTTTCATTATGCCTTGTAACTAGTTGCCTTGTTATATAAAACTATATATATATATAT ATATGTATGTACATCAGTATTATACATATCAATATTTTATATATATATAAAATTGTGTGC TATTTTTGATAAAATTTGTTGTCAGCATTTGTTTACTCTTTTGATGACTTGTATGGCTCC TCCCCATCTTTCCCATGTATTAAATTTTTTTTTTTTTTGAGATGGGGTTTCACTCTTGTC GCTCAGGCTGGAGTGCAATGGCGCGATTTTGGCTCACCACAACCTCCTCCTCCCGGGTTC AAGCGATTCTCCTGACTCAGCCTCCAGAGTAGCTGGGATTACAGGCATGCGCCACCACAC CTGGCTAATTTTGTATTTTTAGTAGAGATGGGGTTTCTCCATGTTGGTCAGGCTGATCTC TAACTCCCGACCTCAGATGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGC AAGAGCCACTGCGCCCGGCCTTGAAATGTTTTTAATAGCATGGAAATGAGGTATTCTGGG AGAAAGAAAGAGAGAATTATCCAGTAAAGTGATTTATATCTGCAAATCTTTTGGAGAGGG ATATAAGGAATTAGTGAAATGGGAAGGAATTCATTTATTAATAGCTTTTTCATTTTTCAT TGGTAATCTTTTTCAGTTTTAAAATTTCTCTGAATATTTTGCTATATTCTTCTTCTCATT TATCATTTTGTTCATATGTTTTCTTTTTTCTGTGATTAGAATTGCCAGAGGCTTGCCTAT TTTTATGTTTTTTTAAACTATATCTTAAATTCCATTTTAAAAACTATTCTATAGTTTTTC TATAACTATGTCTTAAATTCTATTTGTCTTAAATTCTATGTTTTTCTAAAACTATATCTT AAATTCTATTTATGTATTAAATTCTATTTTTATTTTATTAGTATTTTTTGAGACAGAATC TCGCTTTCTTGCCCAGGCTGGAGTGCAGTGGAGCAATCTCTACTCACTGCAGCCTTCGCC TCCTGGATTCAGGAGATTCTTGTGTCTCAGCCTCCCAAGTGACTGGGATTACAGACATGC ACCACCACGCCTGGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTTGCCATGTTGCCC AGGTCTTGAACTCCTGGACTCAAGTGATCCGCCTGCCTAAGCCTCCCAAAGTGCTGAGAT TACAGGCATGAGCCACAGCACCTGGCCTTAAATTCTATTTAAAAATATAATATTTTTGGC CGGGCGCCGTGGCTCACCGCCTGTAATCCCAGCGCTTTGGGAGGCCGAGGCGGGCAGATA ACCTGAGGTCGGGAGTTCAAGACCAGCCTGACCAACATGGAGAAACCGCGTCTCTACTAA GAATACAAAATTAGCCGGGCATGGTGGCACATGCCTGTAATCCCAGCTACTCAGGAGGCT GAGGCAGGAGAATCGCTTGAACCTGGGAGGCAGAGGTTGCAGAGAGCAGAGATGATGCCA TTGAACGCCAGCCTGGGCAACAAGAGCAAAACTCTGTCTCAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAATATATATATATATATATATATATATATATATATATATATATGATATTTT TCCTTCTGCCTTCTTTTATTTTCTTCATTATACATTCAATAGGACATAACATTTTCTAAA ATATGAATTTGAATGTTTAGTTCATGCATTTTCCTTCTTTTTTGTTTTTTATTTTATTAT TATTATACTTTAAGTTTTAGGGTACATGTGCACAATGTGCAGGTTAGTTACATATGTATA CATGTGCCATGCTGGTGTGCTGCACCCATTAACTCGTCAATTAGCATTAGGTATATCTCC TAAAGCTATCCCTCCCCACTCCGCCCACCCCACAACAGTCCCCAGAGTGTGATGTTCCCC TTCCTGTGTCCATGTGTTCTCATTGTTCAATTCCCACCTATGAGTGAGAATATGCGGTGT TTGGTTTTTTGTTCTTGCGATAGTTTACTGAAAATAATGATTTCCAATTTCATCCATGTC CCTACAAAGGACATGAACTCATCATTTTTTATGGCTGCATCGTATTCCATGGTGTATATG TGCCACATTTTCTTAATCCAGTCTATCATTGTTGGACATTTGGGTTGGTTCCAAGTATTT GCTATTGTGAATAGTGCCGCAATAAACATACGTGTGCATGTGTCTTTATAGCAGCATGAT TTATAGTCCTTTGGGTATATACCCAGTAATGGGATGGCTGGGTCAAATGGTATTTCTAGT TCTAGGTCCCCGAGGAATCGCCACACTGACTTCCCCAAGGGTTGAACTAGTTTACAGTTT ACAGTTCCACCAACAGTGTAAAAGTGTTCCTATTTCTCCACATCCTCTCCAGAACCTGTT GTTTCCTGACTTATTAATGATTGCCATTCTAACTGGTGTGAGATGGTATCTCATTGTGGT TTTGATTTGCATTTCTCTGATGGCCAGTGATGGTGAGCATTTTTTCATGTGTTTTTTGGC TGCATAAATGTCTTCTTTTGAGAAGTGTCTGTTCATGTCCTTCGCCCACTTTTTGATGGG GTTGTTTGTTTTTTTCTTATAAATTTGTTTGAGTTCATTGTAGATTCTGGATATTAGCCC TTTGTCAGATGAGTAGGTTGCGAAAATTTTCTCCCATTTTGTAGGTTGCCTGTTAACTCT GACGGTAGTTTCTTTTGCTGTGCAGAAGCTCTTTAGTTTAATTAGATCCCATTTGTCAAT TTTGGCTTTTGTTGCCATTGCTTTTGGTGTTTTAGACATGAAGTCCTTGCCCATGCCTAT GTCCTGAATGGTAATGCCTAGGTTTTCTTCTAGGATTTTTATGGTTTTAGGTCTAACGTT TAAGTCTTTAATCCATCTTGAATTAATTTTTGTATAAGGTGTAAGGAAGGGATCCAGTTT CAGCTTTCTACATATGGCTAGCCAGTTTTCCCAGCACCATTTATTAAATAGAGAATCCTT TCCCCATTGCTTGTTTTTGTCAGGTTTGTCAAAGATCAGATAGTTGTAGATATGCGGCGT TGTTTCTGAGGGCTCTGTTCTGTTCCATTGATCTATATCTCTGTTTTGGTACCAGTACCA TGCTGTTTTGGTTACTGTAGCCTTGTAGTATAGTTTGAAGTCAGGTAGCGTGATGCCTCC AGCTTTGTTCTTTTGGCTTAGGATTGACTTGGTGATGTGGGCTCTTTTTTGGTTCCATAT GAACTTTAAAGTAGTTTTTTCCTATTCTGTGAAGAAAGTCATTGGTAGCTTGATGGGGAT GGCATTGAATCTATAACTTACCTTGGGCAGTATGGCCATTTTCACGATACTGATTCTTCC TATCCATGAGCATGGAATGTTCTTCCATTTGTTTGTATCCTCTTTTATTTCATTGAGCAG CGGTTTGTAGTTCTCCTTGAAGAGGTCCTTCACGTCCCTTGTAAGTTGGATTCCTAGGTA TTTTATTCTCTTTGAAGCAATTGTGAATGGGAGTTCACTCATGATTTGGCTCTCTGTTTG TCTGTTGTTGGTGTATAAGAATGCTTGTGATTTTTGTACATTGATTTTGTATCCTGAAGA CTTTGCTGAAGTTGCTTATCAGCTTAAGGAGATTTTGGGCTGAGACAATGGGGTTTTCTA GATATACAATCATGTCATCTGCAAACAGGGACAATTTGACTTCCTCTTTTCCTAATTGAA TACCCTTTATTTCCTTCTCCTGCCTAATTGCCCTGGCCAGAACTTCCAACACTATGTTGA ATAGGAGTGGTGAGAGAGGGCATCCCTGTCTTGTGCCAGTTTTCAAAGGGAATGCTTCCA GTTTTTGCCCATTCAATATGATATTGGCTGTGGGTTTGTCAGAGATAGCTCTTATTATTT TGAGATACGTCCCATCAATACCTAATTTATTGAGAGTTTTTAGCATGAAGGGTTGTTGAA TTTTGTCAAAGGCCTTTTCTGCATCTATTGAGATAATCATGTGGTTTTTGTCTTTGGTTC TGTTTATATGCTGGATTACATTTATTGATTTGCATATATTGAACCAGCCTTGCATCCCAG GGATGAAGCCCACTTGATCATGGTGGATAAGCTTTTTGATGTGCTGCTGGACTCAGTTTG TCAGTATTTTATTGAGGATTTTTGCATCCAAGTTCATCAAAGATATTGGTCTAAAATTCT CTTTTTTGGTTGTGTCTCTGCCTGGCTTTGGTATCAGGATGCTGCTGGCCTCATAAAATG AGTTAGGGAGGAGTCCCTCTTTTTCTATTGATTGGAATAGTTCAGAAGGAATGGTACCAG TTCCTCCTTGTACCTCTGGTAGAATTTGGCTGTGAATCCATCTGGTCCTGGACTCTTTTT GGTTGGTAAGCTATTGCCACAGTTTCAGAGCCTGTTATTGGTCTATTCAGAGAGTCAACT TCTTCCTGGTTTAGTCTTGGGAGGGTGTATGTGTCGAGGAATTTATCCATTTCTTCTAGA TTTTCTAGTTTATTTGCATAGGGGTGTTTGTAGTATTCTCTGATGGTAGTTTGTACTTCT GTGGGATCGGTGGTGATATCCCCTTTATCATTTTTTATTGCTTCTATTTGATTATTCTCT CTTTTCTTCTTTATTAGTCTTGCTAGGGATCTATCAGTTTTGTTGATCCTTTCAAAAAAC CAGCTCCTGGATTCATTAATTTTTTGAAGGGTGTTTTGTGTCTCTATTTCCTTCAGTTCT GCTCTGATTTTAGTTATTTCTTGCCTTCTGCTAGCTTTTGAATGTGTTTGCTCTTGCTTT TCTAGTTGTTTTAATTGTGATGTTAGGGTGTCAATTTTGGATCTTTCCTGCTTTCTCTTG TGGGCATTTAGTGCTATAAATTTCCCTCTACACACTGCTTTGAATGTGTCCCAGAGATTC TGGTGTGTTGTGTCTGCTCTCGTTGGTTTCAAAGAACATCTTTATTTCTGCCTTCATTTC GTTATGTACCCAGTAGTCATTCAGGAGCAGGTTGTTCAGTTTCCACGTAGTTGAGCGGTT TTGAGTGAGTTTCTTAATCCTGAGTTCTAGTTTGATTGCACTGTGGTCCGAGAGACAGTT TGTTATAATTTCTGATCTTTTACATTTGCTGAGGAGAGCTTTACTTCCAACTATGTGGTC AATTTTGGAATAGGTGTGGTGTGGTGCTGAAAAAAATGTATATTCTGTTGATTTGGGGTG GAGAGTTCTGTAGATGTCTATTAGGTCCGCTTGGTGCAGAGCTGAGTTCAATTCCTGGGT ATCCTTGTTAACTTTCTGTCTCGTTGATCTGTCTAAGGTTGACAGTGGGGTGTTAAAGTC TCCCATTATTATTGTGTGGGAGTCTAAGTCTCTTTGTAGGTCACTCAGGACTTGCTTTAT GAATCTGGGTGCTCCTGTATTGGGTGCATATATATTTAGGATAGTTAGCTCTTCTTGTTC AATTGATCCCTTTACCATTATGTAATGGCCTTCTTTGTCTGTTTTGATCTTTGTTGGTTT AAAGTCTGTTTTATCAGAGACTAGGATTGCAACCCCTGCCTTTTTTTGTTTTCCATTTGC TTGGTAGATCTTCCTCCATCCTTTTATTTTGAGCCTATGTGTGTCTCTGCACATGAGATG GGTTTTTTCCTGAATACAGCACACTGATGGGTCTTGACTCTTTATCCAATTTGCCAGTCT GTGTCTTTTAATTGGAGCATTTAGTCCATTTACATTTAAAGTTAATATTGTTATGTGTGA ATTTGATCCTGTCATTATGATGTTAGCTGGTTATTTTGCTCATTAGTTGATGCAGTTTCT TCCTAGCCTCAATGGTCTTTACAATTTGGCATGATTTTGCAATGGCTGGTATCGGTTGTT CTTTTCCATGTTTAGTGCTTCCTTCAGGAGCTCTTTTAGGGCAGGCCTGGTGGCGACAAA ATCTCTCAGCATTTGCTTGTCTGTAAAGGATTTTATTTCTCCTTCACTTATGAAGCTTAG TTTGTCTGGATATGAAATTCTGGGTTGAAAATTCTTTTCTTTAAGAATGTTGAATATTGG CCCCCACTCTCTTCTGGCTTGTAGAGTTTCTGCCGAGAGGTCTGCTGTTAGTCTGATGGG CTTCCCTTTGTGGGTAACCCGACCTTTCTCTCTGGCTGCGCTTAACATTTTTTCCTTCAT TTCAACTTTGGTAAATCTGACAATTATGTGTCTTGGAGTTGCTCTTCTCAAGGAGTATCT TTGTGGCGTTCTCTGTATTTCCTGAATCTGAATGTTGGCCTGCCTTTCTAGATTGGGGAA GTTCTCCTGGATAATATCCTGCAGAGTGTTTTCCAACTTGGTTCCATTCTCCCTGTCACT TTCAGGTACACCAATCAGACGTAGATTTGGTGTTTTCACATAGTCCCATATTTCTTGGAG GCTTTGTTCGTTTCTTTTTATTCTTTTTTCTCTAAACTTCCCTTCTCGCTTCATTTCATT CATTTCATCTTCCATCACTGATACCCTTTCTTCCAGTTGATCACATCAGCTCCTGAGGCT TCTGAATTCTTCACGTAGTTCTCGAGCCTTGGCTTTCAGCTCCATCAGCTCCTTTAAGCA CTTCTCTGTATTGGTTATTCTAGTTATACTTTCGTCTAAATTTTTTTCAAAGTTTTCAAC TTCTTTGCCTTTGGTTTGAATTTCCTCCTGTAGCTCGGAGTAGTTTGATCGTCTGAAGCC TTCTTCTCTCAACTCGTCAAAGTCATTCTCCGTCCTGCTTTGTTCCGTTGCTGGTGAGGA GCTGCGTTCCTTTGGAGGAGGAGAGGTGCTCTGCCTTTTAGAGTTTCCAGTTTTTCTGCT CTGTTTTTTCCCCATCTTTGTGGTTTTACCTACTTTTGGTCTTTGATGATGGTGATGTAC AGATGGGTTTTTGGTGTGGGTGTCCTTTCTGTTTGTTAGTTTTCCTTCTAACAGACAGGA CCCTCAGCTGCAGGTCTGTTGGAGTTTGCTAGAGGTCCACTCCAGACCCTGTTTGCCTGG GTACCAGCAGCGGTGGCTGCAGAACAGCGGATTTTTGTGAACCGCGAATGCTGCTGTCTG ATCATTTCTCTGGAAGTTTTGTCTCAGAGGAGTACCCGGCCGTGTGAGGTGTCAGTCTGC CCCTACTGGGGGGTGCCTCCCAGTTAGGCTGCTCGGGGGTCAGGGGTCAGGGACCCACTT GAGGAGGCAGTCTGCCTGTTCTCAGATCTCCAGCTGGGTGCTGGGAGAACCACTGCTCTC TTCAAAGCTGTCAGACAGGGACATCTAAGTCTGCAGAGGTTACTGCTGTCTTTTTGTTTG TCTGTGCCCTGCCCCCAGAGGTGGAGCCTACAGAGGCAGGCAGGCCTCCTTGAGCTGTGG TGGGCTCCACCCAGTTGGAGCTTCTCGGCTGCTTTGTTTACCTAAGCAAGCCTGGGCAAT GGTGGGTGCCCCTCCCCCAGCCTCCCTGCTGCCTTGCAGTTTGATCTCAGACTGCTGTGC TAGCAATCAGCGAGACTCCGTGGGTGTAGGACCCTCCGAGCCAGGTGCGGGATATAATCT CCTGGTGCGCCATTTTTTAAGCCCGTCGGAAAAGCGCAGTATTGGGGTGGGAGTGACCCG ATTTTCCAGGTGCCGTCTCTCACCCCTTTCTTTGACTAGGAAAGGGAACTCCCTGACCCC TTGTGCTTCCCGAGTGAGGCAATGCCTCACCCTGCTTCGGCTCATGCACGGTGCGCTGCA CCCACTGTCCTGCATCCACTGTCTGGCACTCCCTAGTGAGATGAACCCGTTACCTCAGAT GGAAATGCAGAAATCACCCATCTTCTGCGTCGCTCACGCTGGGAGCTGTAGACTGGAGCT GTTCCTATTCGGCCATCTTGGCTCCAGCCTGACTGCATTTTCCTTCTTGTTGTAAATAAT AAAAATATTTATAGGGACAATGCTTAGGGGTACAGCTTTGTCTCTGTCTTGTAGGTTTTA ATATGTGGCATTTCAATTTTTAATATTTTCTAAATGTCTTTTGTATTCTTATTTTCCCTT CCTCTTTGGTTCAGTTTCTTGCAGATTCTTCTTTAAGGATGTTTATAGTAACATTTGTGG TATCTGACTTGGCAAGTTGCAAGTATTGTACTACATTTCCCACATTAGTTACCCTTGTAG CTGTACTTTTCCTTTCAGATCCTCTAGTAATTCAAAGAGACCCACTCAGGTTCAGGCAAA TATAAGAAGCTTCTTGTGAATGGAATGGCTTATCTAATATTTCTAAAATGACTGGAGCAA TGACTTACTGTTTCTAAGGCTTGAGGGACTTGGGCGCAGAGGCAAAAAATCACTCCCGCT AGAGGTATGATGGTGAAAGCTGGCTAGCAGCTGTTTGTACCTCTGGTGGGGAGTAGGACT GGGGACTCCCAGACCTTGCAGGCACTTATTAGGGAATACAACATCATCAGGAAAGGGACT ATGTGAACAAGGTTGGATGCTCCAAGGAAAAATGGATTGATGACCCTGGGAGGCTTTCCT CCTGGAATCTGGGCAATGCCCACTCTCGTCATGCTTCTTTAGAACATGTTCTTATTTTCA AATGTTTTCATAGATTTTAAATGGGAATTTGGGAAGGCAGTCTCCAGTCACTTCCACCCT ACCTTTTTCCTGGAAGTGTTCTCTAAATCATGTACACATCTATTCTCCTTTACTTGGCTC TAGTGGTTTCTGGGACACCGGTTAGTGGTCAGACTTTGAAAACTGCCCTTTCTTTGACTA CAAGGTCTGGTTTGGTCTATATGTCTCTGGAGAATTCTTCTCCCTTCTAGGTGGTGTCTT TGTAGGGACTGTCTGTCTCTGTTGCTGGGTGGAACCTGGAACCTGGTTAAGGCTGGCCAG TGGAGGGGCTGTGCCTTGAGGAGGGCACAGATAGCCCCAGGAATAGGTGCCATCCTGGCA ATGTCCTTGGGGAATGTGGTCTCCCCTTTGGAGATTTGGCTGTTGCATGAGGTGCTAACT GAGTGCAGGGGTTGCTACTCGTTGCTTACTGCAGCTTCCCTGGAAGCCTTTGATGAGGAG GGCAGGACCTATGGAGGAGACAGATCAGGAGAGTGCATTGTTTCCACAGCTGCGGTTTGC TCTGCCTAGCCCTTGTGGTGAGATGCAAGGAGCCTTCTTTCTCTATTATGGGAAGGTAAT TTGCTCTGAGGCCATTTTCACTATTAAAATACCCATAAGGCAGGGAGGAAAACCCCTCTT TAAGCAGAGAGGCAGGAGTCAAGTGTGGGGAAAGCTGTTGCTCTCCCCTGGAAGTTTTGG ATGAACAGTGGATACGTGAATGTGGGGTGGGAGGAGGCTGTTAAAAATGTCTTCATACAA GGAGGCATGTAATTTCCTCCTGGTGGGCATGGTTTTACTCCACATCAGATGTCATTTTGA TCTGCCTGTCCCATCAATGGTCAGGCTTCAGCTGGAAAAGAGAGAATGGTGGAAGTAAGA GGTGGGAGACTAGAATGTCAGGCAGAAACAGCAGCGACAGTCTGCTGTCTCATTTGCTTA AGATCTGTATGAAGACTTGAAAAATCCCACAAATCTGCAATTGCAGATTTTTATCAAAGC ATCTATAACTTTATAATTTAAAACTGGTATTAAAAGATGTTGCCTTTTTAAACTCTTTTA ATGCCTGTTTATATGAGGACATTTTTAACAACCTCCTGCAACCCTACATTCACACACACA TATTTTAGGACAGACATTATTTAATCTGATATGCTCATTTGTCACACAAAAGGTGAGCAG CTCAGAAAAAGCCTGAAGCAAGGAGGAGCTCTTGCCTGTAGCAGCCTCATGACCATTTCT GCTCTTGGACTTGGCTGCTTTGAGTTTTTTCAAGTCCTCAAGGCCTTTGTTCTTTAGCTT TCAGGGCTATTATGACTGGATAGTTTACCAGAAAAGGTCCACTTCTTTTACTCTGAACTG ATAAGCTGTTTAACTGAATTGCTTGTTTCTGTCCCAAGTGAGCGTGTGTATGTGTTTGTG CTTGCAGACTCAGTATTTTTGCAGCTTGGGATTATAACAAAAGATACGAGATTTTTTTTT TGAGACAGCAGATAAAAAGGGAAATAAATTAGAGAATTATCTTAGTCAATTTTATGTTGT GATAACAAAATACCACAGACTGGATAATTTGTAAAGAATACAGATGTATTTCTTACAGTT CTGGAGACTGGGAAGTCCAAGATCAAAGGGTCTGTATCTGGTGAGGGTCTTCTTGCTGCA TCATCCCATGGCAGAAGGCAGAAGGGCAAAAATGTGCACATGAGAGAGCAAGGGTAAAGA GGCCTGAACTCACCTTTTATCAGGAACCCACTCCTGAGATATTGGCATTAATACATCACA AAGGAAGTGTCTTCATGGCTTAATCACCTCCTAAGGGTCCCACCTCTTAATACCATAATA ATGCCAATTAAATTTCAACATGACTTTTGGAGGGAACATTCAAACCATAGCAAGGATAGA ATATGATGGAAGATTCAGGGAAGAGAAAGGAAGGGCCCAGTTTTTATTAAAATTGTGACA AAGTACACATAACATAAAAGATACCGTCTTAACTATTTTAAGTTTATTGGAATTAAGTGC ATTCACAGTGCTGCACAACATTATCATTATTCATCTCCAGAACTCTTTTCATCTTGCAAA ACTGAAAGTCTATATCCATGAAATACCAACTCCCCATTTCAATCTTCCCCTCAGCCCCTG CCTCATATAAGTGGATCACACAGTATTTGTCTTTTTATGACTGGATTGTTTCACCTTGCA TAATGATTTCAAGGTTTATACATGTTGTAGAATGTGGCAAAATTTTCTTCCTTTTTCAGG CTGATTAATATTCCATCACATGTATATACCACATTTTACTCTGAACTGACAGGTTGTTTG ACTGAATTACTTGTTTCAGTCCCAAACATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGCGTGCACAGACCCAGTATTTTTGACAGTGTGGGAATATAAAAAAGATGTAAGCT ATTTTCTTTTTTTAGGAGATTGTCTACTTATCTGGCAAAGGGCATTTGGGTTGCTTCCAC CTCTTTGTTATTATGAACAGTGCTGCTATGATCATGGGTAGGCAAATAAGTTCTTGAGAA CCTGCTTTCAATTCTTTTATACATATACCTAGAAGTGGCTTTGCTGGATCATATAGCAGG TCTATTTTTAATTTTTGAGGAGTCTTCATACTGTTTTCCATAGGGATTGCACCATTTTAC AATCTCATCAACAGTGCACAAGTGTTCCCATTTCTTCACATCTTCATCAACACTTGTTAT TTTAGGTTTTTTAAAAAATAGTAGCCATCCTAATGGGCATGAGGTCATCTCTTTGTGGTT TTGGTTTGCATTTTTCTAATGGTTAGTGATGTTGAGCATCTTTTCATATGCTCGTTGGCC ATTTGTATTTCATCTTTGGAGAAATATCTATTCAAATCCTTTGGTCATTTTTTAATTAGG TCATTTAATTTTTTATTGTTGAGTTGTAGGAGTTCTATTTTATTTTATTATTTAATTTAA TTTTATTTTACTGTAAGTTCTGGGATACAGGTGCAGAACATGAAGGTTTGTTACATAGCT GTATGTGTGCCATGGTGGTTTGCTTCTCCATCAACCTGTTATCTAGGTTTTAAGCCCCAC ATGCATTAGGTATTTGTCCTAATCCTCTCCCTCCCCTTGCTCCCCACCCCCAACAGGCCC CAGTGTGTGTTGTTCCCCTCCCTGTGTCCATGTGTTCTTATTGTTCAACTCACACTTAGA GTGAGAACATGCAGTGTTTGGTTTTCTGTTGTCAGAGTTATTTACATATTCTGGATGTTA ACCCCTTGTTAGGTATATGACTTTCAAATTTTTCTTCTATGAGTTTTATGTTTTTCAGGT CTTACATTTAGGACTTTAATCCATATTGAGTTAATTTTTTATATGGTGTAAGTAAAGATT CTAACTTCATTTTTTGCAAGTGGAGATTCAGTTTTCCCTTTACCATTTGTTGAAAAGACT GTCCTTTTTCCAAGGACATTGCCAAGACTATTCCATAGTCTTGTCACCCTTGTGGAAAAT AATTTGGCCATATATGCAAAAGTTTACTTCTGGGCTCTCTATTCTATTCCGTTGGTCTAT ATGTGTCTTTATGCTAGTACCACCTTGATATGGTTTGTCTGTGTCCCCACCCAAATCTCA TCATGAATTGTAGTACCCATAATCCTAACGTGTGGTAGAGGGCCCTGGTGGGAGATAATT GAGTCACGGCGAGGGTTTCCCCCATGCTATTCTCGTAATAGTGAGTTCTCATGAGATCTG ATAGTTTCATTAGGGGCTTTCCCCTTCGCTTCACTCTCATTCTTCTCCTTCCTGCTGCCG TATGAAGAAGGACATGTTTGCTTCCCCTTCCACCATAATGGTAATTTTTCTGAGGCCTCC CCAGCCATGCTGAACTGAACTGTGAGTCAATTAAATCTCTTTCCATGATAAATTACCCTA TTTTGATTACTGTAGCTTTGTAATAAATTTCGATATCAGGAAGTGTGAGACCTCACAATT TGTTTTTTTTCAAGATTGTTCTGGCTATTTGGGGTCACTTGAAATTCCATATGAATTTTA GAATGGATTTTTATATTTTTGTAAAAAATGTCATTGGGAGTTTGATAAGGATTGCACTGA ATCTGTAGATCACCCTGGGTAGTACGATATCTTAACAATATTAAACCTTCCAATCCATGA ACACAGGTATGAAAAAATTCAGCTATTTGCTTGTCATTTACTCTCTTCATTGTTCTGTCA CCATTGTCTCCTTCTGTACATCAGAGTGACAGCTCTAGAATGGTGGTGACAAAGTACAAC CTCCAGTGCTTGGGTCTCATGTTACCTGAAACTATTCTCAAGAATATGGACATAAAGCTA GGCCTTGAAATGCAGCTTCACAATTTATCTCTGGGCCTGTTTCTCTGAAATTCTAAATAC CAGGACTTGCTTAAAAAGCTTGCATTTTTGAACTCTGGTTTGCTGAAGACCAGAATGTTA AATGTCACATTGAAATGGAAAAAAAAATCAAGGCCAGTAAGATAGAAGGGCTCTTTGCTT TTCACATTGTTTATCATGTTTCTTCCTGTTGTAAATGATTTGCTTTTTAGGTGTGTCTTC ACTGGGATTTATATTTTTGAAGCTTTGATTAAAATATTGGCAAGAGGTTTCATTCTGGAT GAGTTTTCTTTCCTTCGAGATCCATGGAACTGGCTGGACTCCATTGTCATTGGAATAGCG TAAGAATATTAAAGATGTTTTTGAAGAGACTTGGAGTGGGAAAGAAGCCCCTTTGCTTCC ATCAGAGCTCTGTGTGGATCCATTTGAGTGGCTGAGTATGGCCTAGTAATGCCTAGAGCT CTGAGTGCTGTGTTGGTTGGCCCTGAACAGCACGTGCAGCCAATTGGCCAGAGGCCCCCA CACACAAAGCCATTTCTAGGTCAACACTCAAGTTCGTGTTCCTTCAGCCATTTACTCACC AACACTGGCCTGTTATGGGCCAGGCTCTATGGAAGATACAGGGGTGTGCCAATGAAATTA AAAAAAAAAAAAAAGACATGGTTTTCAATCTTATGAAGCTCACAGTCCAGAGGGGAAAAG TAACAATTAATGGCCCTGAAAATATGGTATGATGAGTACCAGGATAGGGGCTATACATGT ATTAAGCTGTCAGAGAATATAGGAAGAACAAAGCAGTCTAAAAAATCTTTGGCTGCTACA AGGTTACAAAGAGCTTTACTCCTGTTTACTTCTAGAAGTTCTGTAGTTTTGGCTTTTATG TTTCAGTCCATGGTCCATTTTGAGTTAATTATCATGTATGGTGTAAGGTGAAGGTTGAAG CTCTTCCTGAAGGAAAGACACTGATTTGGAATCTGAATGATGAAATGGAGTCAGCAAAAG AAAAAATAAGAAATGCAGTGACACATTGCCAGCACAACAATAGTAAACCTGGCATCATTC AATATGTACCAGGCATTATGTTAAGCACTTTGCATATTTTTTCCTATTTAATATTCACAA AAGCCCTTGGAGGTAGGTGCTAGTAATTTCAGAGTTTTACATTTGAAGTTCAAAGATTGT GTGCTCTTCGTTATTGAAAGGTGGTGAAGCACAGTGGTTTAGAGTATGGCTCTGGAGCTA CACTGCCTGGCTACACATTCAGGCTTGACCATGTAGTGGCAGGAGACTTTAGTGAAGTGC CTTGGGGCATGGAACTTGTCAGCCACTCAGTAAATATTTGAGAGGACTTCTATTATTCAT GGATGTAGCCACAACTACATGAACTATTTCAAGGAGTCTTCATCTGTGTCCACGTATGAA GGTGAACTGAGAACAGCGTCCTCTGCAGTTCATAGCAAGAACAGTGGTATTGCCAGATCC TAAGATAAAGGTGGGGCTGGAAAGGTGAGGCTGTCGATTTCACCTTGGAGGTCACAGGCC TCAGGCTGAGCTTACTTGTGGCTTTTGTGGAGATGTGCAGTGCAGCTGCAGGACTGAGCA AGCCCATGCCTTCCCTGTGCTTTGTCTTGTAGGATTGTGTCATATATTCCAGGAATCACC ATCAAACTATTGCCCCTGCGTACCTTCCGTGTGTTCAGAGCTTTGAAAGCAATTTCAGTA GTTTCACGTAAGTCACTCTCACTTTCCACACTGGCTCCTATCTAGAAAGAATGTTAATTT ATATTTAAATTTTTTCAAAATGTCATCATGTAAACCAAAAACAAACAAAGCCTGCTTTAC AAATTTATTTGATGGTCCAACTGATAATTATTGAGCACCTAATATGTCCAGGCACTGTGC TAACTGCTTGAGATCTAGTAGTAAGCAAATGGATACGGACTCATGGAGTTTTACCTTCTA TGGTGGGGGTGGGGAGACAGACACGAAGCAACAACTATAATAAATAAGTACATTATGTAG CATGTGAGCAAGTGCTATGTGCCAGGGAAAATATAAAGAAAAAGTAGACACCATGAGGGC TATGGGAAGTCACAGGGTACAGGGGAAGGAGGTTGCAGTTTTAAACAGAGTGATCACGAT AGTTGTCATTGAGGTGAGAGGTGGCAGAGATTTGAAGGTGGCAAGGGAGTGACACAAGCA GATATATGGGAAAGAGTATTCTGAGCATGGGAAACAGCCAGCACAGAGATTGAGGAGTAG CAAGGATGAGGATGGGACTGGAAAGGAGAGGGTGAGAGGCCAGGTAGAGGTGGGATCATA GAAGTGAAGGGGGGGCCGGGTACAGTTGCTCACGCCAGTAATTCTAGCACTTTGGGAGGC CAAGGTGGGGAGATAACTTGAGGCCAGGAGTTTCAGAACAGCCTGGCCAATATGGAAAAG CCTCGTCTCTACAAAAATACAAGAGTTAAGGCCAGGTGCAGTGGCTCACTCCTGTAATCC CAACACTTTGGAAGGCTGAGGCAGGTGGATCACCTGAGGTCAGAAGTTTGGTATCAGCCT GATCAACATGGAGAAACCCCATCTCTACTAAAAATACAAAAGTAATCAGGTGTGGTGGTG CATGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGTGGGAGAATCACTTGAACCTGGGAG AGGGAGGTTGCGGTGAGCCTGGATCGCCCCATTGCACTCCAGCCTGGGCAACAAAAGCAA AACTGTCTCAGAAAAAAAAAAAATTAGTTAGATGTGGTGGTGAGTACCTTTAATCTCAGC TACTCGAGAAGCTGAGGCAGGAGAATTGCTTGAACCTGGGAGGCAGAGGCTGCAGGCTGC AGTGAGCCAAGATTGTGCCATTGCACTCCAGCCTAAGGTCTAAAAAAAAAAAAGGACTGA AGGGGAGTCTTGTAGGCACAGGGAGGATATGCGCTTCACTCTGAAGAAATGGGAGCTGTT GTGTGGAGAATGGAATGTAGGGGAGATGGGAGAAGCAGGAAGACCTCCTGCTACAGGGAG CACAGTGGACTAAAGATCCCGGCTTCCACCTCTCTGGATTCCCCACTGCATTCATCCACA TGCCAAGATCATGCCTGCCCTGCACAGTGTGTGTGGGTTCTAGTGCTGGCCCATTCCTGA AGCATATGGGATTCTTCTAATAGGAAACTTTGGCTCAGGGACTCTCCAGTGACCTGGCTG AGACTCCGACAGCTGCACTGCAGTCTGAGGTTCTTCCTATCCAATCCTCCTCCCTTGTCT GTCTCTCAGACATCAAACTTGAGCTGCAGTCTGAAGTTTCTCCCTGCCCACTCCTGCTCC CCCGACTCATCTTTCACAGGCGTCCTCCCAACCCCCACAAATCTCTGGCACATCTGATCT CAGAATGTCTGCTCCTCAAAACCCAAACTGACACAAGAGACCATAATCCAAGCAAGGGAT GGCAGTGCTCTCTGATCAGGTAGTGGTAGTGGAGTTGGTAGACACTGGTTTGATTCTGTG TCAATTCAAAGGTAGAAACAATAGCATTTCCTGACTGATCAATTGTATACATGGTGTGAG CAAAAGAGAGGAGTCAAAAATGACTTTTATACCTGATGTCACTGATTGAGACAGAGAAGG CTGTGAGAGGAGCAGGCCTTGGGGTCAGATTACAAGCTAGCTTTGAACATGTTATGTTTG AGATGGCTGTGAGACCACCAAGGGAAAGGATTCAGCAGGAAATTAGGTATATATGAGTCC AGGAGTCCAGGGAGAAGTCAGAGCTAGAGATACCATTTCAGGAATCAAGGGTCTATAGAT ATTTAAAGCCGTGAGATCTAAAGAGATCACCAAGGGAATAAGCACTGATGGAGAATAGAA GAGGACCAAGGTCTGAGCCCCAGGGTACTGAAACATGAAAGGGAGAAGAGGAGAAACCAG GGAAGGAGATGAGCAGCAGCCACCTGTGAAGTAGGAGGGGAACCAAAGGTGGTCTCCTGG AAGCCAGGTGAGGAAGTCATGTTAGCCCTTCGGATCAGGATGAAGCCAAGGTTGGGTGTG AATATGAATTTCTGTTTATTAAAAATGAGGTTATTATAGCACACCCTTTTCCTCTCCTTC CCCTTTCCTCTCTTTTCTTTACTAAAACAAACAAACAAAACAGTTTATTATCTTCCCTCT CTGGACTCCTTTCCCCCCACCTTCTCCCCTTCCCTTCTTTTCTCTTCTATCAGTGGCTTT GGCAAACTGTAAAAAGACATGGGAACTATTTAGTTGTGCTGATGATATAGACATAAACTA TACCGCATGGCCCCAATACCTAAACTCCAGTCCTTTCAAACAGAAGCAGTAGGTTATTTT TTGAAACTTCAGAAAAGCAACTGAATAGTGATAAAGAAACTGGTAGTCAGAGGACACTTT ATATACAATTTATCTCTTCTTCTCCTCCTCCTCCTCCTCCTCCTTCTCCTTCTTCTTTTC AAGTAGGACTTTGTTTTCCCAGGTAGATTTAGGAGCTTTCTGCATGAGCTGAGCCCCTTT TATTTTCACATAAGTAAATACACATTCAGGCAGTCATTTAGCAGCTTTCATGTCCCCGTC TTCATCTCCACACAGGTGAATATAGTCTCTATTTTGGCCCATTAAATGCACTGAGATGCC TGGATGCTTTTTGGAAGTAAGGGTGTGAGGCACAATCTTTTATGAGACTATTAAAAAATG ATCCAGGAATCACAATCATCACTCATCCACATTTCTTGCTTTGGAGAAGCCTCTTGTTAT AGATTAAAATCTGCCTTGGGTGACACTGTAGTAACCTCTGGTTTGCCTTCAATTGGTGAT ATAGTAAGTGTCCCAGCTTGGCCTCCAGGTTCCTGCTATCTGGTGTTATACTTTGTGGGA AGCCCATCCACTGAGGGCTTGGCCTCCCACTGTATGCTGCCATGTGCTCTCACCTCTATG CTAATCAATTGAAATTTTGTCACTTAGTGTCATTCCATGCCTTTCAATTACATCTGGGAG TTTATCTCTCCAGCTATCTTGAGACTCAGTGGATTGCTGAAGTTGACTTGTTCTTTTTCC AGCTCTGTCCAGTGACATCATATTGGTAGCTTGAAATCTACCATGGTGGTGAGTGGGGAT CAGGATCTATACCACAGAATTCAGAAAACAAGGAAACCTGTAAATGCTATAGATCAGGGC TCTCCTGCCTCCTGCTTGAAGAGCCAGTTTTAAATATTTACTAGCATGCCACAAGATTAT TTATTTTGTAACTTTGGCAGCTTAACAGTGTGCCATGGACATAGTAGGTGCTTAATAAAT ACTTCTTGAATGAATGAATGAATGAAAAGAAAACACCCTATTCTAACTGGTAATCTGAGT TTTCCCTCCCAGGATATCCTCATAATTTGGGTCTAGACAAATCTCCTTCATCCCCTATAC CCCACAATTTAGCTGTGTCTCATGGTACTCAAGTACTTTGACAGTAGACAGTATCTACTA AATGTTAAATACAGGTCTATCTCATTCTCTAGTGTGGCCCCAGCTTAGGTGGTGCCTTTG CAACTGTCCTCAACCTATGCCTTGAGTCCGTGTGGCATGTTTCTATTCAAAGCCTCTTTG CTTGCTCCTCATAGCAGCACTGAGGCAGGCATAGGGCAGGAATAGAGAGATTGAGTCTCA AACTTGTTGACTGCTTTGATGACACTCAGCTTCCAAGGCACAGAGTTGGAGCACAAACTC AAGGCATCAGAATCATAGGCTAGGCTGGAGTCCAGTTCCAGCCTGCAATTTGACAGTTTC CGAGACTTCAGTTGCTCCTTACTTGGCCTTTGTGAGTGTCTGGCTGAGGGGGATGGGCTT CTCCCCAAGCCTGTCCTCTTTCTCAGGTCTGAAGGTCATCGTGGGGGCCTTGCTACGCTC TGTGAAGAAGCTGGTCAACGTGATTATCCTCACCTTCTTTTGCCTCAGCATCTTTGCCCT GGTAGGTCAGCAGCTCTTCATGGGAAGTCTGAACCTGAAATGCATCTCGAGGGACTGTAA AAATATCAGTAACCCGGAAGCTTATGGTAAATACCCAACCTTTCTTTCACTCCTTGGTTT TTGCATGGTTAACTTTTCCTTATCCTCACTTGTCCTGCCTTCACACTTCCCTTACAGAGT CTCTTAGCAGCAAGGGAGGACTTCATCCATCCAGGGTGCTCCTGTGGGCAGATGCATTTC CTAGAGAATTGCCTCATTCTTTGGCTTCCTATGGAGAAGGAAAGTCTTGAAACCACATCC ACAAGAGCAATTCTATGCTGTCCCATCCCTTTGCCATTTAGATCTCTGCCCACTTCTTTG TAATGATTTGCAGAAGTTTTTAGAATGCTTGGATTTGGCTAACAATAATCAGCAGCTTCC CAGCCCCACTCCTCAAGTATTTCTCCAATGACACGTCTTTTTTTTTTTTTTTTTTTTGAG ATGGAGTCTAGCTCTGTCACCAGGCCGGAGTGCAGTGACATCACCCCACTGCAACCTCTG CCTCCCAGGTTCAAGTGATTCTCCTGCCTCAGCCTCTCAAGTAGCTGGGATTACAGGCAT GTGCCGCCACACCCAGCTAATTTTTGTATTTTTAGTAGAGACGAGGTTTCACCATGTTGG CCAGGATGATCTCCATCTCCTGACCTCGTGATCTGCCCACCTCAGCCTCCCAAAGTGCTG GGATTACAGGCGTGAGCCACCATGCCCGGCCAACAACACTTCTTTCAGTGCCCACCCTCT CCATGCTGAGGTTGACCCTACACCTACTCCATGTGTCATGAATCTCATGTGACTACACTC TCTTGATGTCTCTCATGGGATGGGTCTGTGTCCAGCTTCCCCCAGGGAGGATCTTCGGTT CAGTATTATTTAAGAGAGGCAAAGTGGTGGTGGTGATGTCACTGCTTCCTCCTCCAACTT CTCCTTCCTTCTTCTTTTCCCTCCTTCCTTCTTACTCCTTCCAGAGGTCTTGGGCTCCAG TTTCTCTCTGGACACACCTCCTTTAATCATTTTAGAAGATGTAGAAACATCCATTTTCAA GCATAATCTGCATACTTACTATTCTCTTTCCTTTCAATGTTTTTTTTAAAATCTGAATTT ATGACTTTTTCCTCTCTCAGACCATTGCTTTGAAAAGAAAGAAAATTCACCTGAATTCAA AATGTGTGGCATCTGGATGGGTAACAGGTAAGAGGTTTATAATCATTTTCCTCCCAAGAA GAGTACCTCTAGACCTTTTAGAATGGTGTGGCAAACAGTCTTTAATGACTGGTGGACACA GTGATGATAGTTCTCACATAATTATTTCATGTGTCTAAACATGTTTAGACTTGTGTTATT AAATTAAAAAATCATTTCCTGGTGTTTAAGGCAGCTTTCCTCCCTAGTTATGTTAAATTG GAAGCTTATAAGAAACCTACTGAAACAGTGATATTTCAACTTTATGGAGTAGGTATTCAA GACTTTCAAAACTGAGAAGGGATGTTATGACTGAGAGCCCTGTGAGGCCCTTGTTTCCTG TTCAGTGGCATTGCCCTGCTGCAAGAGTATCTGAGCATGCTTCTTCTCTTTGTTGACCTG TTGACAACTATTGCCTCAAATTACAAACACAGTTGAACATACATCCACTTACACTTGCCC CAACAGCTGCTGGCCCTCTGCTCCAATGGGGAAACTCTTTTTTCCTTCAGGTTAGCGGTG CTTTCTATTGTGAAAGATTCAATTCAGTGAAGATTTATTGAGTGTATTTCAAATTAAGAT AATATGCAAGGTACTCTGATGAAATAGATGACATTCATTCCTAAATTACAAATAGATAAC AAGAATCTACAGAGTGCCAAGCCCTGAGTTTGCCTCAAGGAATGCAATTGTGAACAAGGC AGCTCCACGTTTTTGGATTTGATGTCTTCTAGGGAAGACAGATGCACAGAAAATGCAGTT ATGTGTTGCATAATGATATTTAGATTGATAATGGATGATATATATGATACTGGCCCCATA AAATTATAATACTATATTTTTACAGTGCCTTTTCTGTGTATACACAAATTATTACCATTG TGCTACAGTTGCCTACAGTATTCAGTATAGTAACATGCTGTACAAGTTTGTAGCCTAGGA ACAATAGGCTATGCCACATAGTCTAGGTGTGTAGTAGGCTATACCATCTAGGCTTGTGTG TACACTCTATGATGTTTATACAACAAAATTGCCTAACTACACATTTCTTAGAATGTATTC CTGCTGTTGAGCAATGCATGATTACTAGAAAGTGTGATGAGTGTTATAAGGGTGATGTGC AGGTGCTGAGAGGTATGATGGGGTTGTGACCTTGTCTGGAGAATTAGGGAAGGCTTCTCT CATTCCATTTGCAAGACACGATAGGCTTTAATCTAATATGAAGCTTACTGAGCTTTCTAG AATGATTCCCAACAGCACACACTGCATTGATATTGTTGAGGATCATTCTAGAAAGAAAAA CTGTCTTCCATGAAACCGGTCCCTGTTGCCAGAAAGCTCCCCAACATTTTGGCACCAGGG ACTGATTTCATGGAAGATAATTTTTTCGTGGGCAGGAAGTGGGGGTGGTTTTAGAATGAA ACTGTTCTACCTCAGATCATCAGGCATTAGTTAGATTCTCATAAGGAGCGTGCAACCTAG ATCCCTTGCATGCGCAGTTCACAATAGGGTTCACACTCTTATGAAAATCTAATGCTTACC ACTGATGTGACAGGAGGTGGAGATCAGGTGTAATGCTCACTCGCCTGCTGCTCACTTGCT GTACAGCCTAGTTCCTAGCAGGCCATGAACCAGTACTGCTCTGGGGCCCGGGTTTGGGGA TCCCTGTCTTAAAGGGAGCCCGACAGCTGCTGCATATAGCACTTCTTTTACCCCTCACAC AGGCAGACAGGTGTGGTTACCATAGGCAGAATCCAACGTGGGGTGTATAACCTAGACAGG CAGAAGCAGCAGCCTTGTTTCCAACTAGTCCTTTTTTTTTTTCCCAAGTCTTTTGCCTCC AAAATCTCTTATTACAATACTATACTGCTATAAAAAGTCCAAAAAACTATTTTAGCTGTT GAAAACTCAAATAACATCAGACACAAAATGAATCAAGTGTGATGTGTATATGACAATAGA GAGTGGTGGGGAAAGTAGAGAGGCAGAGGACACATACCATGCTTAACAGCAGTCAAACTT GGGTATTTTTTTTATTTCAGTAGGTTTTGGGGGAACAGGTGGTACTTGGTTACATGAATA AGTTCTTTAGTGGTAATTTCTGAGATTTTGGTGCACAAACTGGTCTTCTTTTTAGCTTTT ATTTTAAGTTCAGGGGTACATATGCAGGTTTGTTATATAGGTAAATTTGTGTACAGATTA TTTCATCACCCAGGTATTAAATCTAGTACCCATTAGTTATTTTTCCTGATTCTCTTCCTT CTCTCAACCTCCACCATCCAATAGGTCCCAGTATCTATTGTTCTCCACTGTGTGTCCATG TGTTCTCATCATTTATCTCTCATTTACAACTAAGAACATGTAGGATTTGGTTTTTCTGTT CTTGCATTAGTTTGTTAAGGTTAATAGCCTCCAGCTCCATCCATGTTCTTGCAAAGTAAT AGAACTCATTCTTTTTATGGCTGCATAGTATTCCATGGCACATATGCACCACATTAGCTT TATCCAGTCTGTCATTGTTGGGCATTCAGGTTGCCTCCATGTCTTTGCTCTTGTGAATAG TGTGACAATGAACTTTCACTTGCATGTGTCTTTATGATACAATGATTTATATTCCTTTGG GTATATACCCAGTAATGGGCTTGCCAGGTCAAATGGTAGTTCTGTTTTTAGGTCTTTGAG GAATTGCCACAGTTTTCCACAATGGTTGAACTAATTTACACTCCCACCAACGGTGGATAA ATGTTCCTTTTTCTCCGCATCCATGCCAGCATCTGTTATTTTTTGACTTTTTAGTAATAG CCATTCTGACTGGTGTGAGATGGTATCTCATTGTGGTTTTGATTTGTATTTCTCTAATGA TCAATGATGTACAACTGGTCTTTATAGGGCTCTTGTCTCTAAGTTTATGTGCCAGCACAT CTCTCTCAGTCTAGGTACAATATGAATGCTCCAGAGTTTTGTGGTAAACCAACTCCTAGT ACACAGCATTCCACAGTGATCACAACATGCTTGCTTCTGAGGAGATGGCACTCTGAGAAG GCAGGTCTGTGATCATCTTTCCAGGCAGGCTTTGGTCTTTTCAGGGCCCAGGCTAGTCTT CACCCTCCAGTCACTGCTTCCTCCCCTGCTGACTAATTCACCGTGACTACAACTCAGATC TGAAGTCTAAGTAGGAGGTTCCAGTAGATAAAGAGTGAACAATTCCAGACAGTAGGCACA GCAAGGGTGAGCATAAGGCCTTGGAGCACAAGAGCATATAGGGATTGACAAGAAGCCCAG CAACTTTTTGGGCAGACAGGCAAGAGAGCAGGAAGGAACAGAGACAAGAGCCAACCATGA AGGACGTGATAGCTCATCAGTGCCAACGATTTTGGTCTTGATCACAAAGGAAGCAGGAGA CATGGAAAGGTTTTAGGGCAGGCAGATGACTGGGCAGATTAGACATATTTTAAAAGCCAC TATGGTTTCTGTATAGAGAATGGATGCATAGAGTAGATGAGGAAGCCAATTACAAGACTG TGGTAGAAAGAATAAGGATCATGAAGATGTACAGTGGTGATGAAGAAAGCTGGATGGATG CATTCCAGAGACGTTTAGGAGACAGAAAGACTAGGACATATCAATATTAGCCTTGAATGT CAATGGGCTAAATGCCCCACTTAAAAGGCACACAATGGCAAGCTGGATAAAAAAGCAAGA CCCAATGGTATGCTGTCTTCAAAAGACTCATCTTACATGTAATGACACACATAGGCTCAA AATAAAAGAAATGGAGGAAAATATACCAAGCAAATGGAAATCAGAAAAAAAGCAGAAGTT GCAATCTTAATTTCAGACAAAACAGACTTTAAACCAACAAAGATAAAAAATAGATAAAGA AGGGCATTACATAATGGTAAAATGTTCAATTCAACAAGAAGACCTAACTAATCTAAAGGT ATATGCGCCCAAGACAGGAGCACCCAGATTCAAAAGGCAAGTTCTTAGAGACCTACAAAG AGACTTAGACTCCCACACAATAATAGTGGGAGACTTCAACACAGCACTGACAGTATTAGA CAGATCACTGGGGCAGAAAATTAACCAAGATATTCAGGACCTGAAGTCAACATTGGACCA AATGGACCTGTTAGACCTCTACAGAACTCTCCACCCCAAAACAACAGAATATACATTCTT CTCATCACCACATGGCCCATACTCTAAAACTGACCACATAATGGAACATAAAACAAGCTT CAGCAAATGCAAAAGAACCAAAATTTTACCAAACACACTCTCGGACCACAGCACAATAAA AATAGAAGAAAAGACTAAAAAAATCACTTAAAACCATGCAATTACATGGAAATTAAACAA CATGCCTCTGAATGACTTTTGGGTAAATAATGAAATTAAAGCAGAAATCAAGAAGTTTTT TGGAACTAATGAGAACAAAGATACAACATACCAGAATCTCTGGGACACAGCTAAGGCAGT GTTAAGAGAAAATTCATAGCACTAAACGCCCACATCAAAAACTTAGAAATAACTTAAACA ATGTCACATCACAATGGAAAGAGTTAGAGAAGCAAGAGCAAAGCTAGCAGAAGACAAAAA ATAAGGAAATCCAAATAAACACAATTAGAAATAACAAAGGGGATGTTACCACTGACCCCA CAGAAATAAAAATAACCATTAAAAACTGCTAAAAACACCTCTAAGCACACAAACTAAAAA TACCTGGACACAAACACCCTGCCAAGACTGAACTGGGAAGAAACTGATTCCCTGAATAGA CCAATAACAAGCTCCAAAATTGAATCTATAATAAATAGCCTACCAACCAAAAAAATCCTG AGACCAGATGGATCCATAGCTGAATTCTACTAGATGTACAAAGAGCAGGTACCATTCCTA CAGAAACTATTCCAAAAAATTGAGGAGCAGAGACTCCTCCCCAAATCATTCAATGAGACC ACCATCGTCCTGATACCAAAACCTGGCAGAGGCACAAAAAAAGAAAACTTTAGGTCAATA TCCTTGATGAACATTGATGCAAAAATCCTCAACAAAATACTTGCAAACCTAATCCAGCAG CACATTAAAAAGCTAATCCACCACAATCAAGTAGGCTTCATCCTTGGGATGCATGGTTGG TTCAACATATGCAAATCAATAAATGTGATTCATCAGAACTAAAGTAAAAAAACACATGAA TATTTCAATAGTTGCAGAAAAGGCTTTTGATAAACTCAACATCCATTCATGTTCAAAACT CTCAATACACTAGATATTGAAGGAACATACCTCAAAATAATAAGAGCCATCTATGACAAA CCTACAGCCAACATCATACTGAATGGGGAAAAGCTGGAAGCATTCTCCTTGAAAAGTAGC ACAAGACAGGATACCCTCTCTCACCACTCCTATTCAACATAGTATTGGGAGTCCTGGACA GAGCAATCAGGAAGAAAAAGAAATAAAGGGCATCCAAATAGGAAGAGAAGAAGTCAAACT ATTCCTGTTTGCAGATAACATGAATCTATATCTAGAAAACCCCATAATCTCAGCTCAAAA GCTCCTTCAGCTGATAAACAACTTCAGCAAAGTTTCAAGACACAAAATCAATGTACGAAA ACCACTAGCATTCCTGTATACCAACAGCCAAGCCGAGAGCCAAATCAGGAATGCAATCCT ATTTACTATTGTGACAAAATAATAAAATACATAGGAATACAGCCAACAGGGAGGTGAAAG ATCTCTACAATGAGAATTGCAAAATGCTGCTCAAAGAAATTAAAAATGACACAAACAAAT GGAAGAACATCTCATGCTCATGGATAGGAAGAATCAATATCATTAAAATGGATATACTGC CTAGAGCAATTTACAGATTCAGTGCTATTCCTATGAAATTACCAATGACATTCTTGACAG AATTTAAAAAATGCTGTTTTAAAATTTATATGGAATCAAGAAAGAGCCCAAATACCCAAG GCAATCCTAAGTAAAAAGTATAAAGCTGGAGGCATCACGTTTCCCAACTTCAAAATATAC AACAGGGCTACAGTAACCAAAACAGCATGGTACTGATACAAAAACAGTCATATAGATCAA TGTACAGAATAGAGAGCCCAGAAATAAGGCTGCACACCTACAACCATCTGATCTTGAACA AAGCTGACAAAACAAGCAATGAGGAAAACACCCTATTCAATAAATGGTGCTGGAATAACT GGCTAGCTATATGCAGAAGATTGAAACTGGACTCCTTTCTTACACCATATACAAAAATCA ACTCAAGATGGATTAAAGACTTAAATGTAAATCCCCAAACTATAAAAACTCTGGAAGACA ACCTATGCAATACCATTCTGGACATAGAAATGGGCAAATATTTCATGATGAAGATGCCAA AAGCAATCAGAATGAAACCAAAATTTGACAAATGGGATCTAACTAAACTTAAGAGCTTCT GCACAGAAAACAAAAACAAACAAAAACCAAAAAACTATTAACAGAGTAAACAGACAACCT ACAGAATGGGAGAAAATATTTGCAAACTATGCATCTGTTAAAGGTCTGATATTCAGCATC TATAAGGAAATTAAATTTACAATAAGAAAACAACTCCACTGAAAAGTGGGCAAAGGACAT GAATAGACTTTTTTTTTAAAGAAGACTTACATGCAGCCAACGAGCATATGATAAAAAGCT CAACATCACTGACCATTAGACAAATGCAAATCAGAACCACAATAAGGTACTATTTCACAC CAGTCAGAATGGCTATCATTAGAGTCAAAAAATAACAGACGCTGGCAATGTTGTGGAGAA AAGGGAACACATACACTGTTAGTAGAAGTGTAAATTAGTTCAACCATTATGGAAAGCAGT GTGGCAATTCCTCAAAGACCTAAAACAGAACTACTGTTGCTGGATTCATCCCAGCAATCG CATTACTGGGTAGGTACCCAGAGGAATATAAATCATTCTACTGTAAAGATACACACACAT GCATGTTCATTGCAGCACTGTTCACTATAGCAAAGACATGGAAGCAACCTGAATGCCTAT CAGTGACAAACTGGATAAAGAAAATGTGGTACACATACATGATGGAATACTATGCAGCCA TAAAAAAGCAAGAGATTATGTCTTTTGCGGGAGCATGGATGGAGTTAGAGGCCATTAGCC TTAGCAAACTAAAGCAGGAAACAGAAAACCAAATACTGCATGTTCTCACTTGTAAGTGGG AGTTAAATGATGAGAACTCACGGACATAGAAAAGAACAACAGCTACTGGGACCTACTTGA GGATGAAGGGTAGGAGCAGCAAGAGGAGCAGAAAAAATAACTGTTGGGTACTAGGCTTAG TACCTAGGTGATGAAATAATCTGTACAACAAGCCCCTGTGACATGAGTTTACCTATATAA CAAACCTGCACATGTACGCCAGAACCTAAAATAATAGGTAAAAATAAATAAATAGAAAGA AAAAAATTTGGAGATTGAAGAGGGGATGTTGGTGTGTATTTGTGCATACAAAGCCATGTG TAAGAGAGTGGCGGGAAGTGCAAAGGAGAGAAAACTGTTACAGATGCTTCTCATATCAGT GTGAGTGAGTGATAATGTCATTAGCTGAGATGGGGATGCTGGCGGAGAGGCTTAATCACC TGTATAAGTTGATGAGAGGGTGCACCCAGAGAGGAGGAGATTGAACAAAACAGGCTTATA TTTTGAATGGTGACCAAGAAATGAGGGAGATTAAATATGGTAGGTCCATCCTCAGCAGAC TCCTGGAAGTTAGAGGGCACTGGGACTTCACAGAATTTCCTCCAGGTCTGGTGAGAAAGA GGGCACAATGGATGCCCTCCTCCTCAGTCAACCTAAAGCAAAACCTACCACTCACAAATT CCCACTCTGCCCACCAGGCAAATCCCAATCAGCTTGTCAGGTCCTCTGATTAAAACACAT GAACAGCTCAGGCTCACCAGGCATCTGAAGAAAATCTTTGGTAAAAAGGAGGCCAAGATA AAGCCACAGAAAATGTGGCCATGAACAAAACAAGAGAACCAGAAAGGGCTCTGAAAACTT TGAATAGTGTCCTTGGTGAAATTAAAAGAGATATTATATACATAAAACAAAAAGCAGCTT GTTGTGAAAAAAGAACAGCAAAAAAAGAAACTTATTAGAAATTAAACATTATTACACAAA AATCAACATTAGAACTAAAAGATAAAGTTGACGAAATAGCCCCGAAAGTAGGAAAAAGAG AACAGAAAATTTGAGAGTAAATGTCAATTCAAAAGTCCCAACTTTTGACTAAGTAGGAAT TCTACAACTAGAGCACTGATTAAACAAAAGGGAGGAAATTAGTGAAGAAATGATGCAGAG AATTTACCTGGGACTAAAGATGCAAGTCCCTCAGTGCTCCAGAAAGTACCCAACACAGTG AATGAGAAAGGACCATGCCAAGGACATTATCATGGCATTTCAGAACTTTTTGGAAAAAGA TTTTAAAACTAATGGGCAAAAGAAGGTCACCCAAAAAGGAACAAAAATCAAACTGGCATA AGAATTCTTAATAGCAACACAGGATAATAACTGAGCAGTGCTTTCATATTGCTAAGGAAA AACTTCCAACTCAGAATTCTATTTCTGGCCAAACTACCAATTGATTGTGAAAATGGAATA AATATATATTCATATATGCAAAGATTACACAATCTTCTTAAGAAGCTAATTGACAAGTGC TCCAGAAGAATGAGGGAGTAAAACATAAAAAAGCAACATGAAAAAGCCAAGAAAAAATGT CTACCATAGCAAGAAGTTAATCAACACGAACTACAATTGATAAATCAAATATCAGTGTAA GCACTATTTAGATATACGGAGGTAGAAGAGTTAACAGTTGTTGAATTTGGGGTGTATAAA TGACAGTTAAAGAATTATACAATTGATTTCATTATAAGCTTTTTGGTGCATGTAATTTTT AAGCCATGTGCATGTATTACTTTGATTAAAATACTATTTATTAAAAAACTAAGAAATACA TACCCTTAGTACATCTTATAGATACACTCAATAATTTGTGTTTTGGTAATATGTGAAAAG ACCTATGTTCAAAGATATTCATTGCCCAAATGTTTGTAAGAGTGAAGGACTGTCAATTAC TGAAACCTAAATATTCATCATTAGCAGAGCAGTTAAATAAATCATAGAACTATAGACATG AAAAGAAATAAGATATCTCCCTGTATGGTGATATGAAAAGAGCTCTGAACTATATTGTTC CATTAAAAAAAGCAAGGTGCAGGAGAGTGCAAAGCATGTTCCCATTTTTGTGTATTATAA AAGTTGTGTGTAGATATAGATGTGCTGTATAGTCATAGCAAACTTCTGGAAATTTACAAA AGTAACTGTTAACTATTGCCTCTAAGAAGTGGGATCAGGAAACTGGGAAGAGAGGAAGAC TTAATATTCATTGTATGTGCTTTTATACTATTCATTGTTCACGTGTCACTGTACTCAATT TTTCAATGAAAAACTATCTTATATGAATTTATTAATCAGTTCATTAGCAAAAGAGTCTTG TCAGACAGTGCAGTGGGCCACTTTACAAATACTCCCTTCCTCTAGGGCTCCTGGTGTGAT CCCACAGCCCTTGGAAAAACCAGAGTCGTTGGAAGGAAAAAGAAGCTTGGCTGTGGCGTA CATAATTATTTCTCTGTTTGTTTCCAATATCTAGTGCCTGTTCCATACAATATGAATGTA AGCACACCAAAATTAATCCTGACTATAATTATACGAATTTTGACAACTTTGGCTGGTCTT TTCTTGCCATGTTCCGGCTGATGACCCAAGATTCCTGGGAGAAGCTTTATCAACAGGTTA TCTATTTATAGTCTCTCTTTTTCCTCTCTCCCTCCCTCCATCCTTTTCCTTCCCCCTCTA TCACTTACCTATTTATTTACCATTTATTTATAACTACCATTCATCCCCCACTTTTATCAT CTGTCTCATCTTTAACAGTTGTACTGTTAAATTCTGCCTTACTAATTCTGTTAGAAACTT CAAGAAAATTGGCCAGGCACGCTGACTCCCGCCTGTAATCCTAGCACTTTGGAAGGCTGA GGCTTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCCCAACTCTACTA AAAATACAAAAATTAGCAGGGCATGGTGACAGGTGCCTGTAATCCCCACTACTTGGAAAG ATGAGGCAGGAGAATCACTTGAACCTAGGAGGCGGAGGTTGCAATGAGCCGAGATCACAC CACTGCACTCCAGCCTGGGTGATAGAGTGAAACTCCGTCTCAAAAAAAAAAAAAAAAAGA AACTTCAAGAAAATTTTAAATGCAATATTTGGAAGAAGACTCCAAAGAACCCCTCCTCTT CCTAGAAATGTTAGCCCCCTCCAGATTCCCTACTGCCCTATCTCTCTTGCCACTTTTATT TTAAAAATGCAGATACTCTTGAGAGAGTTATAGATAGTAAAGTCACTGAAACTTCCATGT GAAGGTGTGGATAGTATCTCCCCACTGAGAGATAGGGCATCAGGAATCCCTGGAGTATTA GGGAAGCTATATGGTCTGTGGTCAACTGAGCCCACCAGTGCTGGGGGAAGACCAGTTGTC TGCTACTGTGTGGTTTTTGCTTGAGGTGATGGTCACTTTCCTGTGGAAGAAGGTTTGATA TTCAAGAACATGCTCTGTCCTTTGCAGACCCTGCGTACTACTGGGCTCTACTCAGTCTTC TTCTTCATTGTGGTCATTTTCCTGGGCTCCTTCTACCTGATTAACTTAACCCTGGCTGTT GTTACCATGGCATATGAGGAGCAGAACAAGAATGTAGCTGCAGAGATAGAGGCCAAGGAA AAGATGTTTCAGGAAGCCCAGCAGCTGTTAAAGGAGGAAAAGGAGGTAGGGACATGGGGT CAGACAGTGGGATCTGCTCTGGGGAGGGGAAGGGGTCCCCAGAGGCAATGTGACCTGAGA CTCAAGGCTGCCACCCTTGGCCTGGTCTCAGTGCTTTCTCTACTGGTGCTTTTCTGAGAA TATAAGTCCCTCGAAGAATGGCTGTGCTGCTATCTTTCACAAAATTGTCCAGGCACCAGG CCCTTCAAAACGGGGAACGCTTGGTCTTGATGCTTTTTGACCTTCTGAAGTCTAAGCCTT CTAAGACATGCCCTAAATTACTGACTAGACGATACAACATAAGTATATGTAAATCCAGGC TGTGCTCCAGGCTTTCTTTTGGAAATCTGGAGATGTTTGTGTTCGGCGCCTTTGCTTGAG GTGGACTCTAGAATAATGTTCACCTCTCCCTGTCTGCAATCCTTGTCTTATTTATAGGCC ATTCTAATAGTTTCCTTGGAATCTTTACATTCTTCCAGCTCAAGTCTCAGATTTTCAGGA CAGGGACCATGTTTTATACTCGCACTGATCCTCTATAAGATTTGTCAGAAGGATGCCCAT CGTAAGTGCTTAACATCTGCACTATCAGGCATAAGTATTTATTGAGTGCCTAGCGCATAC TGGACACTGTGCTAAGGGGCAGTGACACAGAGATATGAAAGAGTCAATGCTGCTTTTGGT GGCTGCAATTTCTGTGCAAAAACAGGACATGTTTCCCCAGATAAATAACAGAGCTAGCGT AGTCCAGGTAGATGATGAGCAATTTAAATGATACAGAGCTACCAGAAGAGAGATTTTCAC CACTCTCAGGTCGGGGTCATTTGCAATGTTTCATGAGGAGGTGTAATCTGTTCTTTAATT GCTTTTGTTCAATTTGAAGGCTCTGGTTGCCATGGGAATTGACAGAAGTTCACTTACTTC CCTTGAAACATCATATTTTACCCCAAAAAAGAGAAAGCTCTTTGGTAATAAGAAAAGGAA GTCCTTCTTTTTGAGAGAGTCTGGGAAAGACCAGCCTCCTGGGTCAGATTCTGATGAAGA TTGCCAAAAAAAGGTAAGTCTTTTCCAGGGAATTAATGATACCATATTGCTGTCCAGGGG GACAGGTCCAATTGCCAATCAAATTGAAATAATTCATTTATTCAGTTACTCATTTATTCA TTCATCCATTTGCACATACTTTCATGCATCCATGTGTCTTTTCAATTATCCATTTATTTG TAAACATCAATTTAGAGTGTTTACAGTGTTTACTATCTTCTTGGCACTGGGATAGGTGCT AGGGATACATGTTTTATACTCCACAGAGTGTACGGCTAATGATGAAGATATACTGATAAG AAGGCAGTTATGAGTCAGTGTGATGACTGATGTGCTGATTGCTGTGGGAGAACAGAGGAG GAACTATGTGTCAGTGTGGGCTCCCTCTGAAGCAAACCCAAAGACATTACAATGCAAGTA GATTATTTGGGAAGTGATCCCAGGAAGCACTGGGAAAGGAGGGAAGAAGTGCTATATGGA ATGGAAGGTAACCAATGAAGATGCATTATCAAGCCAGTTACCACTGAAGCCAGGTAGTTG GAGTTTAATCCTTCCAAAGTAAGGGCATCAAAGTGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTATATATAGATATATACACACACACACACACACACACACACACACACATATATGTTG GTATATATATACCAACTTTGGACCATCATAGATTAAAGATTGCTGAAGACAGAAGACAAT AATTTCCCTCTAATTTCCTACTTGCTGTGCACTAAGGAAGACAGGATTTGAATCATTAGA GAAAGTCCTCAAGTAAGGACATATGGGTGGGAAAACCAACAGCATCCAGAATTCATGGGG CCTGGGGGAAAAGCAATGGAAAAGGGTCCCCAGAAGATGCAGTGTGTCAGCTGAAAGGAT GAATTCACCAGTAAAAGGAAAAAGAGAGCATTACCTGCAGAGAGAATAGAATGTGCAAAA GCCTAGAAGCGAAGGCAAGCAGGAGAACATGACCCATTAGAAAACTTGCCAGTGTGTGTG TATGCGCGTGCACGCACGCTCATGCATGCACATGTGTGATGAGGCTGGGGGAGGGTGGGT GGCACAAGGCTATAGTGATCAGTTGGGGGATCACAAAAGACCTCGAAAACAAAGCTAGAG AGTTTACCCTTTTCAATGAGGGCTAAGGGTAAAGAGCATTGAAAGGTTTATATAAGAGAG GGATATGACTGGATTTATGAAATTCTGGATGCATTGAAGACAGGGGATTGGAGGAGGGCA AAACTGGAGGCGACCAGTTAAGAGGCTGTTGCAGTAACCAAGTAAGAGGTGCCTGTGGCT TAAACCAGGGTTAGGGAAGTGAGGATGAAGAGCAATTAACAGACGACCGTGATCTGGAGA AGATAAAATTTAGCAGTTGACTTGATGGACAGTGTGGGGATAAGACCTGGTTTCCGACAT GATTAGCTAACTTGATGGAGGGTAATTTATGGAGACAGGAAAGGTACACATTTTGGGAGT GGAAAGATGGCTCATTCAGTTTTGAATGTGCCAATTTCCAGATGCTTATGGGACCTCCAA GCAGGGATGTTGATTAGGCAGCGTGATGTGCAGAAATGAAGCTGGTGGTGGGGGAGGGAT GGGCAGCAGAAAAAGTTATTCTTATCATTAAGGAGGGGGATGTTCAGACATATTTTAATT GTCCAGAGAAAAGTCAGAAGAGAGGAAGATGCTAAAGATACAGGTAAAGATGGCAAGAGC CACTTTCCTGAGATAGAGGGAGATGTAGTAGGTAGAAGGTGGATATGTATGCCTGATAAT GTGGTGGGGAAAGGGAATTGAGAGGCCTTTGCAGAAGTAGGTAGAAGGATCCCATCCACT GCTATGATCAGTTCTCTGCTGTAGTAATAGGGAGAGATCTCATAGAAAGACTATCTAGAT TGGCTTATCACAAAAAAGATAGTTCCCTCTGGGGCTCTGGTGAGTCGGAACCCTTGGAAA ATAAGCCACTAGGAAAACCCAAATTTTCTGGATAGCTGGAGGTTTGCACTGTTTAATTCC AGTAACTTTAGAATTGTAGGTAGATGTGAGAGAGTGAGCTAGCCCTTGGGCCCAGCTTGA GAGTGTGTTTGAGGTTTAGGATCTAGATTTTGTGATATTTAGTGGGACTGTTCTTTTTAG TTACCAACCGTTTTAAAAACCGTATAGGCGGTTGTTCACTTCACAACTGCAGCTAGCACT TCACAATCTGGCTCCAGTCCACCTTTCTAAATTTATACTCCACTTCTTACCTTTAAACAT TGTTTACAAATTGGATTTTGTATTGCTTTCATACCCGGAACCATTGCTTACTAGTTTTTG TAAAATTTTGTTTGCTCTTTGTTGGCAGTGAATGAAACACAGAGAAAGTAAAATGTCTGT AGGTAGGACTCTAGAAATGTGCATGCATAGGAAATATGAATGATCAAACCAGATTTCTCT TATTTGTTAAACATGAAATAAGAAAACTGAATATTTGGCATGCTGTAAAGACGAGGTGAG TAGACCTATAGAAAATGTTGAGCACTTTCATTACATGTAGCATTGGAACAAAGTAGTTTG TTAAGTTTTGAGGAGAGGCAGCCCCTGTCTTTAGAAGGTGCCCTATGCCTTCTCTTACAG CCACAGCTCCTAGAGCAAACCAAACGACTGTCCCAGAATCTATCACTGGACCACTTTGAT GAGCATGGAGATCCTCTCCAAAGGCAGAGAGCACTGAGTGCTGTCAGCATCCTCACCATC ACCATGAAGGGTAAGTTCCACATCCCAATCCAAGGGAAAGTCTACTTCAGTGATGTCCTT CCATTCTTCTTCTTCCCAATCCCCTAGAAGCCCTCTGCAAAGGAGGCTGTTTTGGAACCA ACCCACTGTACCATCCTGCAAAGAGTGACTTCCTTTGCCTCTTTGGAGCATGTAAACAAA GTGGCTCACAAGAAGCAGACCTTCCCTGTGGGCCCACTCTAAACTGGCACCTGGGTAGAG GTGGAAGCTGGCTAGACTTGGGATGGGACTTCAGACAAAATGCATAAACAGGGCCTCAAA CCAGGCCTGGGAGTCACAAGAGATCCAAGTGACAACAATAGTCAGTCAAAGAACAGAAGA CTTTGAAGTCTCCAGGAGGTGGCAGTTGTTATTGAAGTTAGAGTCAGAAACCCCACATGG GCAATAGAAGACCTATGTAGAAAGGCTGGAGCCATCTGATGGACCAGATTAATAGCAGGT AACAGGACCCCACCCCAGGGGTTGGTGTTCAGGAGCAGGATTTACTCCCTGCAAGGGGGG TCAAATAGGTCCTCTAGCACTTTTAAGCCTACTGCATCATCTCGTTTGCCCTGCATGTGT CCCTGCAGCGGTGCCAGGGAAATCAGGGATGGTCTGAACTCAACTAGAGGATAGAGTCCC TTTTACTGTTTCAGTGCTTATAGATGCCCCTTCCCTTGAGCCCTGGTTGCCCCTTTTGCC TCTGTTACAGCTGACCTTATGGTCTGTCTTGAGTGGACATAACTTGTGCCTGTATCTATC TCTCTCTTGGATTCACAGCCAGCCAATGTTGATTTCCACATCTATATATCTAACATCAAA GCTCAGCTCCTGGATGCTCAGTAAGAGTGTAAATATTTGATGGACTTGACTGTACCGCAT GCTAGTTGTATGTGGAACATTGTCTCTTCTGCACTAAGATTGGTGGAAAAACCTTTCTGA CTACACAAATCAAAACATTTCACAACTGATCATGTATCAGGAGGCCCTGGGGAAGAGGGA GGAAGGAATGCACTCAGGCAAAGGGCATCTGGATTGCTTCAGAGCTAACTCCTCAACCTC TCGCTCATTCTCCTCCAGAACAAGAAAAATCACAAGAGCCTTGTCTCCCTTGTGGAGAAA ACCTGGCATCCAAGTACCTCGTGTGGAACTGTTGCCCCCAGTGGCTGTGCGTTAAGAAGG TCCTGAGAACTGTGATGACTGACCCGTTTACTGAGCTGGCCATCACCATCTGCATCATCA TCAACACTGTCTTCTTGGCCATGGAGCATCACAAGATGGAGGCCAGTTTTGAGAAGATGT TGAATATAGGGAATTTGGTAACATTACACTTTTTAAAAATAAAAAACATATTTCATACTT TAAAGTATAACTTTTTCAAAGTCTAAACATTTTGTGAATGAGCTAACCTAACCTAGGTTT TTGGTCCCCTTCAGTGGAGTGAGGGAAAAGATGGCTGTTACGGTTTTACTTTTACCCGGT TCATTTCAGAAACTGTCCATGTGTCCTGAGTTGGCCCTCTGGTATGTTCAGGTATCTCTT TAGCCATTCAGTGGTAAGCCAGATACAAGCAGACATCCTAAAAGTTCAGCTCTCTGAGAT AATCAAGAAAATGTTAACCATATCCAGAGAACCGTGGTAATGGCTGTTGTGGTCCCAGTG ATATTCATGACATCATTTGTCACAAAATAATGTGACTACTATTACTAGGTGTTATTCTCC CTGGAAAGATGAATCAGGGCCTGTCAGCTGTGGAAGTTTCACTTTTTTGGAAGGGGTTGT TTGGGGGTTGGTCAGGCTGGGGTGGGGTGAATTTCCAGACTTGAGCCTGCTTCAATATGT TTCTCATTGGATAGATCTAGTCTCAGTTAAATTGGGAAAACTAAGGGGAGCAGACTTTTC AAGGACTGTTCACTTCAGTTTATGGTTCTTGTGGAAGGCATTTTTCTCAAGGAAAAATTA GTCCCTCGAATATCAGAAACAACAAATAATTGCCCACGGGATGATCTACCCTAATTAGCT GATGGATTAATGAGCTAAAAACCTTACCAGAATGTCTAGCCTTTTCTTCTTAAACTTTTG TTTTATCTTTCTAATTTAAAAAATAATAAATGCTGTTTTATTTTTTAAAAATAAGGAAAA ATATAAATAAGGGAAATAGAAGAAATTCATGTCCCATGTACCTGCTGACAAAGATGATCA TTTTTAGCATTGCAGAATATTTGCAAGCTTTATGTAAGCATGTATGTATGTGTGTATGTA TTTTTATTTAGTGTTATTTTTTCTCCTTTTTTGGCATTTTTTTTTTTGTGATGTCTTTTT ACATGTCTTTAAAAAATTTGTTTTACATTGTTAAGAACCCACTGCAGCCGAGCACAGTGG CTCACGCCTGTAATCCCAACACTTTGGGAGGCCGAGGCAGGCAGATCATGAGGTCAGGAG ATCAAGACCATCCTGGCTAACACGGTGAAACCCCATCTCTACTAAAAATACAAAAATTTA GCCGGGCATGGTGGCAGGCGCCTGTAGTCCCAACTACTCAGGAGGCTGAGGCTGGAGAAT GGTGTGAACCAGGGAGGCGGATCTTGCAGTGAGCCGAGATCGTGCCACTGCACTCCAGCC TGGGTGACAAAGTGAGACTCCATCTCAAAAAAAAAAAAAGAACCCACTGCACTAGAGTCA GGTACCTGTCATCCTTAGTTCCACATCCCTCCACAAGGAGCAGTGATACAGTGGAACTGT AGTATAAAACCAGGGTGGTTCTCCTCACAGGCAGTCTGCACATAGCACATAAATTCCTTT CTCCATGCTGCCTTTGGGAATAAATGCTTTGTGGGTGTGAATGGGTTAGACTAGATTCCA TGCTGCTTGACAAACCTAACACATAGTTGCTTGTAACAGATCAGGGTCTCCTGGCCATGT ATTCAGCTATCCTCTGCTGAGGCATCTCAGAAATGAGCTGAAATTGTTTCAGTTGAAGGC AGCAAGCTGTGTCTATAGGGGCATGGTCAGCAGACTCCACCTTTTAGCACCTCCTGGTGG AATGGAGTTACTGAATAAGTTGTAGAGAATACTCAAAAACCCAGAGTCATCTCTGTGGAC AGGGCTTCCCAATTGCAGTGGTGATCTGTTTTCATCTGCTGGTCCAAATTTAGAAAATGA GAAAAACCCCCATCTCAGATTTACCACTGACTAGACATCCTGAGTGTGGAGGTGAAGCTA CATTTTGCAGTTGGAAACTGAGTGATTGACATTCTTCTTCACTTCCTCCAGAGCAGGAGG AGCCACAGGGCCACAGGGACATTGTTGTTTTCCAAGTACACAGGCAGCAGCTGCACCCTA GCTCCTAAAAATTGGCAGGAAATATGTAGAACCATACACATAGCTTGTGTTTCCATTCCA TGGGCCTACTCCCTGGACATACAATACCAAAATGGCAGCCACACTATTTACTGCTCCCAT GGTAGTAGTGGTGGAGAGAAGCCTTGGAGTCTTAAGTTCCAAGGAGCACATCAAAACTTC CCTCCTCTGATGCATGGGCCAGGATTAAGCCACTCTGTAGAGTTCTGCAGGACTGGACTA GAAAATTAACTCTCTAGGGTAGAGATTGCTCTAATCAGAGGGATACACTGAATGCCAAGA AGCCATAAAGAAAAGTTGGATTGAATTGACAAAACAATGGTCCCAAGGTATCTGGTGATG CTGTAGAGCTTGATGCCTAAAGCCTAAAGGAGATACAAGAAGGAGGAGTGAGTCAAAAGA GGTGGGGATGACTACACAATTATCTAGGACAGGCATCACTGTATGCTTTTAGATACTGGT GGTGACACTGAGCAGGAGTAGATGGCAGTATTTAAAGGGGGAATATGAAAATAAACTTGA ATTTTCTTTGTAACTCTTGATTTATTTTTATTTTTCTACCCTGGATTTATCTTTGCTATC CTTTTGTCTTTAACTTTTGCCATTTTATTTCAGGCATTTTTAAAAATAAATAGTAAATCA TTGAATCATTTATAAAAATAGATAATCCATTATTTTTAATAATCGAATTTAGTCGTTTAT AGGTTCCCCCCAGAATTGTAGTAGACAGTTTTATAACAATCCTTGTAAAGACTTTAATCC TTTTAATATTACCTTATTATTTTTGTATTCTTGCTTTTTTCATTTCTATTTATTTGTGTT TGTATGTGATAAGACTGTGCTGTTTGCCGTGAAGAAATTATTTGCTTTATTTTTTGCAAA GATATGGAAGACAGATTTATTTTTTAATTGGCTACTACATTCAACATTTTTACATATTTC TTAAGCTTGTATTTATCTGATTATTGAAGACAAAGACAAAACTATTTTGCCCATGGAAAA TACAAAATTCAGTACTTTTTGTAATTCCTGCCTCTCCTTTTTATTCTTTGATATTTTGTT CTGGAAATGTGAGCCCTGCATTCTGAATGAGTTTTAGGATTATATTCTGATTCACTAATT CTCCTTTCAACCATAACAAATTTAAGGCTTATCCCATATATTGAGTTGCTCAAAGACTTT TTTTTTTTTTTTTTGCCCCTGGAAGTGTTTTCTAATTCTATGATCTGTGCCCCTTTTTTG TTTTTATTTTTAATTTTTCTGATCAAGGTGTTGAAATTAGGTGGTCAGGTGAACTCCTCC TGGATTCTAGTTTATGGGGGATAATTATCCTTAGTTTTCAGGGCTATTGTTTACATTCCT TCTCTCCCTATTTTAATTGTTTTAATTGGTATTCTAAAGAGAATTCAGAGGAGAAATTGC CTGACACTCTGTCACCCTGCCATCTACCAGTAATACCATTTTGCTTTATCTTCAACAGAA TGAGAAATAATATTCATTTTCCATTCTGGTGTCTGTGAGCTTGGTTACATTGCTCTGGGC ACCATGGAGAGGGATGGAGAGAGGAGCAAAGTCATAGTGAGGTTCATGCTACAATAGTAA ATATTTCTATTGCATGGACTTTAGGGTATAGTGTGTCAATCATGAGAATACTTTTCATTT CATTGTAACCTCTGTACTTCCCTTGGGCCATTCTTAAAAGTGTTATCTCCGCAGCTTCCT TCTCTCATTCTTCTTTGTCCCACTCTAGGTTTTCACTAGCATTTTTATAGCAGAAATGTG CCTAAAAATCATTGCGCTCGATCCCTACCACTACTTTCGCCGAGGCTGGAACATTTTTGA CAGCATTGTTGCTCTTCTGAGTTTTGCAGATGTAATGAACTGTGTACTTCAAAAGAGAAG CTGGCCATTCTTGCGTTCCTTCAGAGTGGTAAGGCACTTTCTTATTTTACTGCATAAACG TAGCTGCCTAAGTGGAAAAAACGTTTTGAGTGAGTTCAGTGGTTATCTTGTACTAAACTT TAATTTATCTTACCTTAACCCCAGAACCAAAACTGCACTCCTCTTCCTGCACAGGTCTTT AATAGTCTTGCAAGTCCCTGGGGGGACTTCGCTTAACTGAACCCTTATACCCTTAGCCCA TTCCTGCCAGAGATGACCCTTTAGGCAACTGGGTACAAAATTGTGTCTCCCAGTGTGAGG ACTCTGTTCCTTGCTCTGTGCACAACCCTTCCGACACTCTTTTTTTCCCATGGAGCTGTG AGGCAATAAATAAATAAGGGAAAATATTCCTATCTTTCAGGTCATATTGGGCTCGCCAAG GGGGGCAAAGACTATAGAATGCCGGGGAGGAATGCAGGGGAGCTGATGGCAGGTGCTATC TATCTCACAAGTTTCCTTCTCACTGCTGCCAGTGCTACATTTCTGTTCTGTAGTCTTTGC CTGCTATCCAGTTTGGAAGCATTCTGGAAACCTTTACAAAGTACCTAATATAAACTGACA TGAGGGAAGGCACAAAGGATGAGGAAATCATTGTCTTTGAGAAGGTTGCAGCACAGGAGT GATACAAGACTGGGCTACATGCACAGTACAGTGTGACAGAGCCCAGGAAAGGTATAGACA GAGGACTACAGAGAAATCCAATCTGAGAAGGGGAACTAGAAAACCTCCAAGGTGGGATTT TGTCCCCTTTCAAAGATATTTACCTTTTTTTTTCAGATAGGATTTTTAAGCCTATAGAGT AAGACACCTCATTCAGTTACCTAAATGTACATTTTATATTTTTTATACTTGGGATATAGT AAGAGGTTACACTGCTCAAAGTCAGGTGTTATTAAGCTTTCCTACTAATGTTTTATTAGC ACTCAGGTAAGCAAAATTGCATTTGCTTTATCATTGTTTAGGCTTATTACAGCCAGGCTC TAATGCACGGAAATCTGTGGCATTGTCTCTGTATGAGAATGGAACCACAGGTTTTTCCTG GGATGCTCCTAGCTCCCCTTTGAGTAGACAATGACAGTCCTCTTCAAGCAGAAGCCTTAC TGACCTGCAACATTTCCATCCCCAAATAGGGGATGTGCTTAACAAAATTGTACTTTGGAG TAAAACCGTCAGTTTATATCAAAAGTTAATATAAGAAACAACCTATAAAACAAGTTGCTA TGTTTTCTCTAAGGTTGTTTCCTATAAAGTCCGTGAAGATCTAGGATAAAATTTCTCTGT GGAAAAAGTATACAAGTCTTGAATAGCTGGTTGCCTTTTCTCTCCTGACTACAAATGCCA TATTACTCTAATCATTCTTATGAGGCTTAGATTCAGACTCGTGGCCTGAATTATATAATG CAGAAGTTAGCAAACTTTTTCTGTAAAGGGCCACATGGTAAATGTTTTCAGCCTTACAGG CTGTGTGGTCTGTGTGGCAACCGTTAAACTTTGCCATTGTAGCACAAAAGAAGCCATAGA CTTTCAAAAACCAGTGAGCATGGCTATGTTCCAATAAACTTTATTTATGGATGCTGAAAC TTGAACTTCATATGATTTTAATGCATCATGAAATATCCTTTTGATTATTGTTATTTTCTT TTTTGAGACAGAGTTTGGCTCTGTTGCCCAGGCTGGAGTACAGTGGCGTGATCTCAGCTC ACTGCAACCTCTGCCTCCCAGGCTCAAGCCATCCTCCCTCGTCAGCCTCCCGAGTAGCTG GGACTACAGACGCACACCACCACGCCTGGCTAATTTTTGTATTTTTTGTAGAGATGGGGG TTTCACCATGTTGCCCAGGCTGGTCTTGAACTCTCCTGTGCTCAAGCGATCACCCACCTC AGTCTCCCAAAGTGCTGGGATTACAGATGAGAGCCACTGTGCCTGGCCTGATTATTTTTC AATCATTTAAATGTGGAAAAGTATTCTTGTCTCTGAGGTCATACAAAAATAGGCAATGGA CTGTATTTGGCTCTCAGGCCAGAGTTCGCTGACGCTTGGTATATTACATATAATTCCAAA ATATTCATTTTATATAATAATTATGTAGCTCAGTCACCATGCTGATCCCAAAGTCCTTAA TGATTAAAAAACTAAAACAAAATATTTCCTTTTTTTTTTTTTTTTATGAGATGGAGTCTT GCTCTGTCTCCAGGCTGGAGTGCAATGGCGCAATCTCAGCTCACTGCAACCTCTGACTCA CAGGTTCAAGCGATTCCCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCATGCGCC ACCATGCCCAGCTAATTTTTTTGTATTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAG GATGGTCTCAATCTCCTGACCTTGTGATCCACCCGCCTTGGCCTCCCAAAGTGCTGGGAT TACAGGCGTGAGCCACTGTGCCCGGCCAATGTTTCCATTTTCAAATAAGAAAGTAGATAC AGGTGAGCATTAGATTATGACAGCGATCTGATGGCTATGTTCAGGATAGATTTAACTTTG AAGAGAGTTTTGGATGTCATAAGTTTGGGTATATGTCATTTGCTTTATAGATGAGCAGAT GGAGTTTGTCCTTAACTGGCTTTTCTTCCATTTTTGTTTGTTGTCTTTTTCTACAGCTCA GGGTCTTCAAGTTAGCCAAATCCTGGCCAACTTTGAACACACTAATTAAGATAATCGGCA ACTCTGTCGGAGCCCTTGGAAGCCTGACTGTGGTCCTGGTCATTGTGATCTTTATTTTCT CAGTAGTTGGCATGCAGCTTTTTGGCCGTAGCTTCAATTCCCAAAAGAGTCCAAAACTCT GTAACCCGACAGGCCCGACAGTCTCATGTTTACGGCACTGGCACATGGGGGATTTCTGGC ACTCCTTCCTAGTGGTATTCCGCATCCTCTGCGGGGAATGGATCGAAAATATGTGGGAAT GTATGCAAGAAGCGAATGCATCATCATCATTGTGTGTTATTGTCTTCATATTGATCACGG TGATAGGAAAACTTGTGGTATGTGTCTTAGATTTTTTTCAAAAAAAAAAAAAAGATCCTA CATGCCTCTCAAATGCATTACTTCATTTGCAAAACTTTACTAGTAAAAATTATTATTCTA AATATAATGCCAAATAAATATTTGAAATATTCATTCAAATTTTGTTTGAGAAAAGTATGA TAAGCATGAAAAAACCTCTTTGACACATATTACTCATTTGACTGATGCCCCCAAATTTAT TTTATTCCAGATGATTATTCTAAGACAATAGTATTCTCTTCCCCAGTGATTGGCTGAAAT AGGAGTTAGTGAGTTAATTTGGTCAATAGGATGTAAAGAGATGTCCACTGGAGCAATTTG GGAACAATCTTTTGTTCTTAAGAAATATGTGTTTTTAAAAAATGATCGTTTTACTTCCTC TGGGCTTTTTTATGACTGTGACCCCTGGAAAAGCCAGATCATGAGCTTAGAGCTGTGGTG GAATTGCCCACAGGAATATGGATCTGACATCCAGGACTGCTGTGCAATCCTCTCCTGTTT GCACGAATTGAGAGATGGTGTACTTTGTGCAGTGTCTTTAGGTCCCTGTTCCCCTAACTT CAATGAGGTATTGGGGCCTAGCCAGAGGGGTTTGGACCAAACCTGCAAACATTATGCAAC CATGAGAAGAGCCAGCCTAAAACAAAGCCAACACTGAGGCAGGTAGAGTGAAAAGAGAGA ACAAACCTGTATTCATGTCATAATTGGCCTGCTGTTCTATTTTCGAACTTCTTGTTATGC TACAGATTTTTGTTGTATAAGCTGGCTTGAGTCTGTTACTTGCAGCTGGAAGCATAGATA TTTATGAGGAGGCTAATTAAGTGCACTTCATTGCCTTACTGCACAAGATCTTCAGGATCA AGTTGGGGTTATAGTGCAATATAATTAACTCTCTACACACGATAGCTTTATCCTAAGTAC CTAATAAATGGTGTGCCTAGACACTAAGTGATACATACAAAAGCTCAGAAGAACAGGGCA TCACCAGTGGGAGTGGGAGGAGAGCACCGAGAAAGGATTCGTGGAAGATTACAGAACTTG AGAAGACCACTTATATGGTAAGGAGGACTTAGACAGATAGAGGAGAGCTTAAGCAAAGAT CCAGAGGTGGGAATTCTCACAGTGCAGCTGAGGAACTGTGACTAGACCCATTTACACCCA CCAGGCACTTCTCTCCAAAGGCTGTCTATTGTCGAGCTGAAGAGAGCCACAAACAAAGGA ATTTGAGGAGGTAAAGATGATGAATTAGAGAGTGTGTAATTATGAGCAAAGAACCCAGGT AGGAAGCTATTGGGATTATGTGAATTTGAAGTGATATGTGGTTCAACGAAAGAGTTGCAT ATTTGGTCCAACTCGCTTGGCTTGGCCCCAGTACGTTACTAAGGTTAGGGCAACAGGACC TAAAGAAACTGTAAAGTACATCATCTCACAAATATTCAAATAAGAAAGGACTGTACATTG GGCTTGAATATCAGATTCATATCCTTGTGGGAAATTCCAGCAAAAATCAAAGCTCACTAT CCAGCTGTTCCTCAAGAAATGTACTCTCAATTTTGCCATATGGTTATCCCATTCTGCTTT ACCCTGGTAAGGGTCTACTCATCCATATTATTTATATGTAGGGAAAGTGGACTTGGCTAT TTGCACCCTTTAATTTCAATTTTTATTCTGTCTTACTGTTTCTATTTCTTTCTCAATCTT TTATTTATTGGATAGATGAGCTTTCTTAGGAGACAGTGGGACATAAAACTCTAATTTGAT TCTGTTTTTAGTCTTGGGGAAAAGTTGTCCTGGAAACCCACTATATCTTTTCCTTTGCTA AACTTTCCTTTCTTCTTGCTACCCACCCCATTCCCAGGTGCTCAACCTCTTCATTGCCTT ACTGCTCAATTCCTTTAGCAATGAGGAAAGAAATGGAAACTTAGAAGGAGAGGCCAGGAA AACTAAAGTCCAGTTAGCACTGGATCGATTCCGCCGGGCTTTTTGTTTTGTGAGACACAC TCTTGAGCATTTCTGTCACAAGTGGTGCAGGAAGCAAAACTTACCACAGCAAAAAGAGGT GGCAGGAGGCTGTGCTGCACAAAGCAAAGACATCATTCCCCTGGTCATGGAGATGAAAAG GGGCTCAGAGACCCAGGAGGAGCTTGGTATACTAACCTCTGTACCAAAGACCCTGGGCGT CAGGCATGATTGGACTTGGTTGGCACCACTTGCGGAGGAGGAAGATGACGTTGAATTTTC TGGTGAAGATAATGCACAGCGCATCACACAACCTGAGCCTGAACAACAGGTATGAAGGTT CACACATAGACTTAAAGGTCATACAAAGCTGGAGTTATCACAGGGCACTGGTAGCCTGCC CTTTTCTAGGCACTATGCAAGGATAATAAGGGTACAAAACGTGGCTGCCCTTTGATACCC ATGTGCACATCTCAGGACAGGTTCTTTATTGTGGTCCTGCAGGTGTGGCACAGATAGGAG GTGTCAGAAGAGTTCAGAGGGGAGCAAAATCAGAGTGGGCTGGGATGACAGGGGGAGTTC CTTTGGTTGAACTCTACAAGAAATGTAGAATTTGGACTGGCTGAAAAGTGAGGAAAAGGG GATTGGGAAGTAAACTGAATAGCATGTTTTGTTTGTTGGTTGGTTTGTTTGTTTTAGAAA AAAAAGGCAGTGCTGGGAACAAACTCAGTTTCTGCAGAGACCAATCTTGTGGGTGAGGAG TGGTGGGAGATGAGGGCTGTCAGGGAGTGTGAGGGTAATGGATATTGTATTAGTCTATTC AGGCTGCTATAACAAAATATTATAGACTGGGTAACTTCTAAACAAGAGAAATTTATTTCT CGCAGCTCTGAAGGCTGGGAAGTTTCAGATCAAGGCAGTGACAGATTTGATGTCTGAGGA GGGCCTGTTTACTGACTCATATTTGATAGCTATAAATTTCTCTCTAACCAATGTTTAGCT ATATCAAATACATTTTATTTTCATTTTTATTTTCTATATATTTAATTATATTTTATTTTA TCTTATTTTCTAATTCTTCTTATGATTTCTTCTTTGACCCACTGCTTATTTAAAAATGTG TTGTTTAATATTTATAAATATCTCAAATTTCTATTTGTTATTAATTTCGAATTTCACTCC ATTGTGATCAGAGAACATACTTTGTACAATTTCAGCCCTTTTAAATTTTTGAGGCTTGTT TTGTGGCATAGTGTATTGTCTATCCTGGAAATGTTCCATGTGTACCTGACAAGAATGTGT ATTCTGCCATTTGGAATTAAGTGTTCTATAGATGTCTGCTAGGTTTAACTGGCTTATAGT TTTGTTCAAATCTTCCATTTCCCTGTTGATCTTCTATCTAGTTGTTATTCATTCATTTCT GAAAATAAGGTATTGAGATCTCCAATGTTTATTGTGAATTGTCTGTTTCTTCCAACAGTT ATGTCAATTTTCATTTCATATATTTTGGGTCTCTTCTGTTAGGTGTGTATATATGTTTAT AAATGCTATATCTTCCCAATATTTTGGTTCTTTTATCATTATAAAATGTCCCTCTTTATC TCTAGTAAGTTTTTTGTTTTTAAATCTATTTTGTCTGATGTTAATAGAGTCATCCCAGCT TCTTTTATGGTTGCTGTTTGCATAACATGTCTTTTTCCATCCTTTTATTTTCAATCTATT TATATCTTTGAACCCAAAATGTGTCCTCTGCAGACAGTGTATAATTGGATCTTGTTTTCT TATCTATTCTGGCAATCTCTGCCTTCTGATTGGATTGGTTAAACCATTCATATTTAATGC TGTTGTTAATTCAGTTTATTTACTTCTCATATTTTCCTGTTTGTCTTCTATATGTCTTCC ATGTTTTTTCCTTTATTATTTCTCTACTGCTTCACTTGCATTAAGTAAATATTTTCTAGT ATAACATTTTAATAACTTTAGTTTTTTCAAAATCTTTTTGAATTATTTCCTTATTTGGGT ACTCTAAAATTTGACATATACATCTAACTCATCAGAGTCTATCTCAGATTTATATTAATT CCAGTGAGCTATAGAAATATTACTCATATATAGCTCTATTTCCTCTCTCCTGTTTTTGTA TTGTTTTAAATTCTACATATTATATTCATATATGTTAAAACCCAAACAACTTTTTTTTAT TATTGCTTAATATAACTAGATGTCTACTGAAGGAGCTGAGAAAATAAAAGAAAATAAGTG TATATTTATAAGATTTATTTTAATAAACTTCTTAGTTACTCTTGGCTGTCTTCATTTGTT CCTGTGACTGTGAGTTACCTGCAGGTGTGATTTTCTTACTCCAATGCAACTTTGCTCCCA CCCATATCCTTTGTGCTCTTATTAGCAAATATATTTTATTTCTATATGTTACATACCCAG CAACACAATTTTAAACATATTGTTTTACACAATTGCTTTTGAAATCAGCTAAAAGAAGAA AGGAAAAGAAATATATATTTTTTATCATACTTGTGTGTTGTATTACCATTTAGGGTCACT TGTTTTTTGCCTGTAGGATGTCCTTTAGTATTCTTTATAAGGTAGGCGAACTACCAAAGA ATTCTCTGTGTTTTTCTGGGAATGTGATTATTTCATCTTCATTTTTGAAAGATAGCCTCA CTAGATATAAAAATCTTGGTTCACTGTTGTTTTTTCTTTCAGCACTTTGAATATATCATA TCACTGCCTTTTGGTCTCCATTATTTCTAATGCAAGGTCAGTGTTAATTTTATCAATGTC ACCTTGTACATGATGAACCACTTTTCTCTTGCTACTTTCAAGATTTTCTCTTTTCTTTGT CTTTCAGCACTTTTACTATGATATGTCTAGGTGTGGATCTCTTTGTGTTTTTTCTACTTG GGGTTTCATTGGGCTTCTTAGATGTGGAGTGCTAGGATTTGAATGTGTCCTCTAAAGTTT ATATGTTGGAGAATTAATTCCCAAAGTAACAATGTTGAGAGGTAAGACCTTTAATAGGTG ATTAGATTATGAGGACTCTGGTCTCATGAATGGATTAATGCTGTTATTGTGGGAGTAGTT TAGTTACTGTGGAAGTGGGTTCCTAATAAAAGAATGAGTTTGCTGCCTTCCCTCCTTTCT CTTTCTTGCCATGTGGTGATGCCTTCTGCCATGTTATGATGCAGCAGGAAGTCCTTCACA AGATACTGGCCTGTTGATCTTGGTCTTCCCAGCCTCCAGAACTGTAGGAAATACATATCT GTTCTTTATAAATTACTAATTTCAGGAATTCTATTATAGCAGCATAAATTGGACTAAGAC GTGTAGATTAATGTTTTCGATCAATTTTGAGGAGTCTTCAGCCATTACTTATTTAAGTAT TTTTTCTGTTCCTTTCTCTCTTTCCTTTCTGGTTGGTGTGCTTAATGGTGCACCACATTT CTCTGAGGCTCTTTTTATTTTTCTTTCTTTTGTTTTCTCTGTGCTCTTTAGATTGCACAG TCTCTGTTGATTCATCTTTAAATTCACTGGTTCTTTCTTCAAATGTACTGTTGGTCTCTC TAGTGACTTTTTCATTTCAGTTATGATAATTTTCAACTCCAAAGTTTCTATTTGGTTCTT TTCAAAATATAATTTCTATTGATATTTTGTATTTGATTGTATATGGTCATAGTATCTTTT ATTTCCTTAAGCATTGTACCTTTTAATTCTTTAAATATATTTATAATGGCTTCTTTGCAT TCTTTTTCTATTAGCTCTGACATCTTGACCCTTTCATGTGCAGTTTCTGTTACTGACTGT TTTCCCCTGTGTGCGTCACACTTTCTTGTTTTTTGCACATTTCATATTTTTGTTTTGTTT TGAATTGGACATTTTAAGTAACATATTGTGGAAACTCTATAACTGATTCACTTTTCTCCT CTCTGAAGCTTCTTTATTAAATGGTAATCTGGCTAGACTATTTTAGTGGAATCTGCTCCC TCTCCAACCTATCCCCCTCCATTCACACACAATGTGAAAGCTTTGGTGTCACTCCTAGGA GAGTACATCCTTGGATGTGCACACGGTCAGTGTAGGATGACTGTTGTTTCAGCAAGGCTA TCTTTGTCTCCTTTTGTGATCTCTCTGTTAAGCTGTCTGCCTCTTTTGGTATTACACTTC GATTTTAGGCTCAACTAATTGCTGGATAATTGCTCTATTTTCCTTGACAATGCCATGGGG CATAAATTGCTCAACTGTCTGATTCAATTAAATTCAGGAAGTGATAGCTTTTAAGCGCAG TCTTTGAGATTTGTTTTGACCCCAGGGGGACTCTCCTTAGCCATCACTTTCCGTAGTTCT TTGTGGTGAATTTGCTGACCTATGTTTTAGTTTCTTGCCTTTAATGAAGGAGAGCTACCA ATCTCTTCTTATTTGCTTATGATCAAAATATCCAATATCCATTGTTTTCAAGAACACTCT TAGACTTATACTTCCCCACACCACTTTGTTTCAAATAAAGTCAGTTCTATGGAGAGCTTC AGAGCTCTTTTTTCTTATGAATTACCTCCCACTCTGGGCAAACTCTTTTAAGCACAATTC CAGGCACTTGGGCAGGGGCAGTAGCCTCTCAACTTCTCAGTTTCCTTCTTCCTTAATCCT CCCCACCCCCAGAGCTTTTGCCCTACAAGTAAGCTGGAATGAGGATGATTCTAGTATTCT TGGGCTGCCGCTTCTGGGATAAAGTCTCTGCCCTATGAGTGGAGGGCTAGATGGAAAAAG GGAGCCAACTTTTTGGCCACACTCACCAGAAATTTAGAGGCCTTAACTCAGAGTTGGAAG AGATAAGAAATGCTGATGGCCTGCAGCTGCTGCTGCTTTTTTTTTTTTCCATAACTCATG CATTTTTATTGGTAGGCATTTCATGGTGGTGTAATCATTTATGCATTGGAAAATGGGATT GTTTTTGATGATACTAAACAGCATTTATTTCTTTATTTTATTTTATTTTATTTTATTTTA TTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTTTTGAGATGGAGTCTCGCTCT GTCGCCCAGGCTGGAGTGCAGTGGCATGATCTCCGCTCACTGCAAGCTCTGGCTCCCGGG TTCCTGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATGACAGGCGCCGGCCACCA CACTCGGCTGATTTTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTAGCCAGGAT GGTCTCGATCTCCTGACCTCGTGATCTGCTCTCCTCGGCCTCCCAAAGTGCTGGGATTAC AGGCGTGAGCCACCGCGCCCGGCCAGCATTTCTTTATTTCAAATGCATTGAATGACCACC ATGTGACAGGCCTTGCACTAAAATAAAATAAGTATGCAATAAGTAATTTTTTTTTTGAGG CGGAGTTTTGCTCTTGCCACCCAGGCTGGAGTGCAACGGCACAATCTCATCTCACTGCAA CCTCTGCTTGCCGAGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTAC AGGTGTGTGCCACCATGCCTGGCTAATTTGGTATTTTTAGTAGAGATGGGGTTTCACCAT ATTGGCCAGCCTGGTCTTGAACTCTGGACCTCAGGTGATCCGCCCACCTCAGCCTCCCAA AGTGCAGGAATTACAGGCATAAGCCACCGTGCCTGGCCTGCAATAGGTAATTTAACATGT AGGCTGTATTTTTTTTAATGTAAGCATTTAAAAAGATTTTCCCCTTATGCAGATATCATT GATGTGCCTTTCCATTCAAAAGGCTCAATATCTTTTTTTTCAATTTCATTAAAAAAAATT TAATTTCAGTAGTTTTTGGGGAATAAGTGGTTTTTGGTTACATGGATAAGTTCTTTAGCA GTGATTTGTGAGATTTTGGTGCACCCATCACCACATCCCTTGATTGAGAGCTAGGGTAGA GGGAGCCCTATCTTCTTGGCCACCTTGGTCTAGAGTGGAGCCTCCATAAAACTTGGCTGG AAGGGATGGGGGGAGAGGTCAGATCATGGCTTAAGTGTCATCAATTTCTGCTGTTCTTAC TGAATTTTAGTAGATTTTCTTGAACAAATGTTTCTTCATTTGCTGTGCACTCTCAGAATA ATTTCTAGAGACTTTAAATTATTTTTAAAACAGTTTTCACCACTTATGCTTATTTCCCTA GGGAGAACGTCCGCAGAGATTATCATGCTATCATCCAGGAAGTGAAACTCTATGTAACTT CTTTATTAAATTCTATATGTATTAATTTAAAGACATTTTCAGATTATTATGTAACTTTCA TCTTTTTCTCAAGTGAATTCACCTCCTAGTTGTTAATTTTAGGAAGTCTACTGATTTTAT GTTTGTTTCCTCATATATTTTGGAATGTTAGCTTAAAAGCTCATCTTTAGTGGGAAGATC TGTCATTCTCTTTGCAGTCTAAGTTCTATGATTGTCTGTAATAAATCCTTCTGTGGCATC CATTTCAGATCCATGTCTTCTATCACCAGCTTTGGGTTGTTGTCTAATAATGATACAAGT CATATTGCAGATCCAGTCACTGAACCAGTGGGTACTTTTGCCCAGGTCCCATTTCTGAGG TTGTACTTCTTCCTGTCTCCCTACGTATGCAGCTTTATACAATTGTAATCATAAGCGTTT GCTGCGACATTTGTGTTTTTTGGCTTTCTCTCTTAAGTGCAAAAACTTTACCTCAGCCCA CAGTTTCAAGCCTTAAATATAGCTCTGGTTCCCTTCCTCATCTAGGGTACTTTTAGACAC CATTACCTGCTATCTTATTTAAATTCTTACTACTTTATATTTCTTTTCTGTGTCTAGTCC ACAGACAGGTTACCCCTTTTTGTATGTGTATGTTTGTGAGGTATATTCGTGGCTATATTT GTATGTGTGTGTATTTTTACATATGCTATTACATGTGTGGAGAAATGTTTCTGAATCATG ACCTTATAATGTCAGAGTTGCAAGAGTCTTCACATAGATGTTAGGATAAAACATTCACCC AAGATGTGACTGTAACCAGCAAAAGGATCCGTCCGGCTGCTAGCTGCTTATGAAAAGAAT TCAAAATAATAACAAATTATGATAAGAAAGAGCAATATTCTCTTATTATCTGTGCCAGCA AGGGGAAGAGCAGAGACCCATTCTACTCTTCAATTTGTGGAAGGAATGCGGGGGTTTTTA TTTTATTTTATTTTTTATTTTTTTATTTTATCATTATTATACTTTAAGTTTTAGGGTACA TGTGCACAACGTGCAGGTTTGTTACATATGTATACATATGCCATGTTGGTGTGCTGCACG CATTAACTCGTCATTTAGCATTAGGTATATCTCCTAATGCTATCCCTCCCCCCTCCCCCC ACCCCACAACAGTCCCGGAATGCGGGGGTTTTTAAAGAAAGGGTTTGAGATGCAGAATAG GCAAGAGGGGCTAGGAGGTGCCAGGTGGTGTGACTTGCTCCAGTGGCCCCTCTTGAATTA TTGTCCCATGTGGTGAAGGGGCTGGTGCCATCATGGATCCTGCCAGGTTATAAATTAGTT GCAGTCAATCTTGTAGTCACTCTACAGCCTGAAGTAGGTTCTGTTCTTGAAGTAATCTTG TTGGGGAGAGAATTCTAGAGGTACCTGGTCCCTATCAGGATCTGACTCTCGAAGCTTCTA AGAAAACATATGACCACATAAAAGAACATGGCGTGTACTTAAAAAGCATTTAGGTAAATA AATGTGCATAAGGTATGGGAATATAGTATGGGAAAGGAAAGGCAGTGGAGGCTCACAGCA CATTCCAAGGCTGTATTTCAAGATAAGAGGTAACACATATGCAGGTTGTCTCAAAGTTGT ATCTTGAGACTTGGAGGGGAAAGAGGAAGAGGAAAAGAAGAAAAAAATGTTTAAAACACA GTTCGAGGCTCAGCTGCCAAGCTGCTTGGTTACATGACTGTACACATCCTTATTAGATAT TCCTGTAGGTCATCAGAACAAATTTATTTTTCAGTTTTATGTAGTGATATGTTTTATTAT TTTTCTTTATGTCTTTTAGTTTCCTGTTTTGCTAAGGAAACTCTCTGTACCCCACATGAT AGCAAGTTATTTTTCAAATTTTTCTTTTAAAATGTTTACCCATTTATTTTTACATCAGAA TGTCATAAGATTATATATTTTATTTTTACTATATTATGGCATTGTGTACCATTTATTAAA TAATTTATTATCTAGCCATTGAATTGAAAGAGCACATTTAAAATATATTAAATCCCCATG TAAATTATATTAAATCTCCATGTAAGTTTTATTTCTGAATTACTTGCGGAGTTTTGTACC AGAACCATACTGTTTTGATTATGTATCTTTGTAACAAGTTCAGTATCTGTTTCCTCTAGT AGTTTTCCCAGAATTATATTATGTTGAGTTTTTCCAGGAGCAGTTTCTAAAGAGAAAAAA GAGTAATAATGTCAAAGGCTTATTCTCATGACAAAGAGAAAACCAAATAGCATTCAAACT TCTTTCATCTTAAGTGACATGTCCAGGAGGAATATTAATTCTATTCTGGGCTCTCTGTGT TAGGCCTATGAGCTCCATCAGGAGAACAAGAAGCCCACGAGCCAGAGAGTTCAAAGTGTG GAAATTGACATGTTCTCTGAAGATGAGCCTCATCTGACCATACAGGATCCCCGAAAGGTA ATTTTCCTAGGATGTTGTCAGAGGAACTTGTGGCTCATCCACACACCTTCTCCAGGATAG TTATTATGGAAAAATGGCACAATTAATGCAGAGAAAATTGCAAGAATCGAGTTTTGAGCA AAAGTCTAGGCCAGGTTTCTCAACTTTACTAGTATGACATTTGAAGCAGGACAATTATTT GTCATGGTGAGCTTTTCTGTGAATTGTAAGATGTTTATCCATATCCTTGGCCTCTATGCA TTTGATGCCAGTAGCATCCGCCAGTTAAGGTAATGAAAAATGTCTCTAGACATTGCCAAA ATGTCTCCTGGAGGGCCAAAGAAATAAGAACCACTGGTCTAGGCCTATTATTCCAGGAGG TGAGACAGTGGAAAATTATAGTCTCAGTAAAGTAGCCTTCTTCTCATCACATAATTGGAG TTAGGCAGCCTATCAGGGTTATGATCTGTAGGGAGAAAACGTGGAGGTACTGTATCTTCA TATGCATTGCTATTCCTCAGCTATAGAGATGAGCTAGAGAGTTCTCTTCTGAAGTCAGGC TGGTCATAGCACTTAGAAAAGGACCCAGCAGGAGGGGTGTTTCTTGACACCTCTCATCCT AGAAACCTTTGCCTAGTTCATTTCATCCTTAAAAAATCAGACAATGAGAAACTCCGTACT ACTATGGTGAAAGAAGGTCTTAGTAAAAGGCACCCCCTTCGTTTTGTTCTGATGTGCAGA AGTCTGATGTTACCAGTATACTATCAGAATGTAGCACCATTGATCTTCAGGATGGCTTTG GATGGTTACCTGAGATGGTTCCCAAAAAGCAACCAGAGAGATGTTTGCCCAAAGGTAAGT GGCAGTATTTCTGCATCCAGTTCACAGAATCTTTGGATGGGGTTTCTGTTCTTCTTTGAA GCTCTGTGGTTTTAGTAGATATCATCTTTCTACTTCTATTGGAGACAAAAGCAATGAGAG GGCACCCGTAGATTCTTGAGGCGATTGTCGGATCTAGGCAGAGGGGATTTCAGGAAATTC TTGCTTTAATTGGGCTCCTCTCACATGGCCCAAAATGCCAGGTAAACACTCTGGAAAACT CTCTTAGAAAGCCCACTGGGCCAATATGTCTGCCACTTTCTATGGTTCCCAGGGCATTCA ATGTCAGGGTGTATTTGAGGTGAAAGACCAAGAAAAGAGTTTAGACATTGTAGTCAGGAT CTTTAGCTTAGAAGCCAGACTGCCTGAGCTCAAATTCTGGCCTTGCCACTTATGAGCTGT GTGACTTGTTCAATTTAGTTAACCTCTCTGTGCTTTATTTCTAAAATGAGGGTAATAATA GTATCCAAAGCAAAGAGTTGTTGTGACTATTAAATGAGTGAATATAAATCACTTAGAAGA GCATCTGGAACAAAAATAAGCAATCAATAAATGCAGGCTTTCCTAATTCCTTGAGTGGAT ATAGTGGCTTTTTCCTATAGGCCTTTACTATTTGTTATATTCATTGTAGTGAACTTATTT GCACTGGTTTCCCCCATGAGGTCCTTAAAAACAAAGGCAGTGACTTCATCAGCATTGTAT CTCTAGTGTAAGAATAGAAGCCGGCCCAGAAATGGGTTCAACAAATGTTGATAAATAAAT AAGTGAAACAAATTAATGAATGAATCTAAAAAAATTATCTATAAGTTTAGGATTAAATTG GGTCCAGAAGCAACACAAATAACAGGTTTCAAACTCACATTCCAGGTTGCTTAACAGCTC CAAAGCTGGACAGGAGTTGGATTAAGGATTTCCAGTAGATTGCAGTTCTCCTCATTTATG TTTGCACTGGCTCTTGAGGTGTGCATATGATGTCAACAGTGGAGATGAGAAAGAAAGGTG GTTTTATCAAAGAAGGGAGGAGTGGGATAAAAGCCAGGAAAAAAGTGTAGTCTTTTAGGA AGATGAACCTACTGATGTTTTTGGAAGAAGAAAAGAGGCAGAGTGAAGAGAGCCCATAGT GGTTTAAGAAGATGAGGAAAGGAGCAGAATGTTATAATCATCATGAATAAGGATCAGCAG AATTTGTACAAGGGGAGGAGGAGAAAAGGTGGAAAGGGCAATGTGGTCTATATTGAAGGC CTGTGTGTGTTCAGAACAAATGCTCAGCTCTTTGTAAACGTCTCGACATTTGATCAAATT CTTTCGTGTATGACCTGAGAGGTTCACAAAGCCTGTGGCCATCCTACCTCATTCCCTGGA GTCCTCCAGACATGATCTCTTTATGATTACCAAAAAAATTGCTGCATAACAAACAACTAC AAAACCTCAGTGGCATAAAATAGTAAACATGTGCCTGCAGGATTCAGCTGATCTTGATTG GCTTCATTCATGCAAGTGTTATCTAGCCTGGGCTCAACAGGGCAACATGGCTCTACTCCA CATGTCTATCATCCTCCTTCCAGACCCAATGGGGTAGCCTGAATTTGCCTTTTTTTATGG AGATGGCACAAGACAGCAAGTGGAAGCATGCAAGGCTTTTCAAGACCTGGGCTCAGAACT GGCACAGTGTGACTTCCATTCCACTCTATTGGCTAAAGCAAGTCACATGGCAGGGACCAG ATTTAAGATGTTAGAAAAAGATGCTCTACTTCTTTAATGGGAGCAACTGCAGGTCGCATG GCAAGGGGCATGAACACAGAAAGGGGTGAAGAGTTGCATTTTAATGCAGTTTATTACAGA CACTAATGATAGTGCTACCATTAATTCCTTTCAGGCTTTGGTTGCTGCTTTCCATGCTGT AGCGTGGACAAGAGAAAGCCTCCCTGGGTCATTTGGTGGAACCTGCGGAAAACCTGCTAC CAAATAGTGAAACACAGCTGGTTTGAGAGCTTTATTATCTTTGTGATTCTGCTGAGCAGT GGGGCACTGGTAAATCATCATTGTCTTGTGGAGACATTGGGCATCAGGGCAGCTGGGGAA GCTGCACTCTGACTTGGCTTCTCGTGGGACTGTGAATATGGGAGACCAGACAGAAGTGGT CTCTGATGCTGCTGGTTCCCGCAGTGGGCAGGGTTTTGCACAGCTTCTGAAATCGTGCTC AACTGGGATATCACCAAGCAAAGATGCTTTATCTGTGTGGAAGACTAACTAAAACCAGCA CTTTAAAGTGGCCACTAGAGTCTGGTCCTTGATCATCTACCTCTCATTTTTGGGGGATTT CTAGACATTTTCACTGTATCCTACATAGTTCTTCCTCTAGACAGCAGTGCTTCTACTCTC CCTGTTACCATAAGCCATAGTGCTGTTTTCTGAAAGGTAAAATATTATGGGAGGGAGTTA CAGGGACCAGATTAGGATATAATAAGTTGCCTTGGTATTTTCTGTCATTCATGATCCCTT GAAAGAAAGGACAAGGAGATGTTGCTTTATATGCATGGTCAAAGTGTGGTATGAGTCCCA ATGAGATTTAGAAATAACTCTGAACCCATCAGCTTCTATAGCCATAGAGCTATCCACTCC TTTATGCTCTCTCTCCACACCTTCTGGTCCTTCTTGTCTCCCTTTCTCCTCCCTGCTAGG GAGGGATAGTATCTTTTCTCTTATCACCTGCATATTTCGCCCTCTCCTTTTCTATTTTCT GCTTTTTTCTATCATGCATCTTCCCTTAAAATGATCATACAGTAATGTGGAGCTAAATAA TAGGGAACCAAATTCTCTTACTGTTTGACATAACAAAGTTATGAGTTTGTTATGAGAGTT TGGTTCCATATTACTAAACCCAGGAGCCTTGGGTTCTCCCCTGCTAAGAGTGACAGAACA TTTCACAGTGAATCACTAGCAAGTGGAAAAAGACAAGGAAATGTCTTGCTCCTACACCCC ATAGCTTCTGGTTAGTGTTTTACACTTGTGTGATCTCTAGGTGAAAAGAGATGAATACAT ACCAGAGCCCACCGGGGACTGTGATCAACATACATGTCCATTTGTCTACTTACTACAGTG TAAATTATTCTTCAAATAGAAAAGCAGATTTCTCTAAAACCATCCCAGAGAATTCCCTTA ATAAATTTTTATTGTTAATCTTGGGTCTAGGGTTACCTGTAGACTTTCTGGTGACTACAT TTTCACTGGCTGTCTACTGTTACTCAAGTGTAGGGTTTCTTGTATTTATTCCACAAATGG TACTTATGATATAGGCAGTGGGAAAGCAGTGACAGTGCCAAAAGGCCACAGCAAAAATGC CCCATGGTGCCATAAAAAGAAAATGTATGATCAGAAATGTAGCAGAGGGCACACAACTCC AGTTCTTATCTGCCTTCGGAAAGGCAGGTGTAGGCTGGGCAGAGCAAAGCTGAACACACA ATGCAGAGGGTGCCAGGGGAAGGACCCAGCCCATATTTCTCTGTACATATTTGTAGAGAA TGAGAACAAGAGACATCAAGTTGTTTGCCACTATATGTGTCTATCAGCAGCAGCAGCAGC ATCACCTGGAAGCTTGTTAGAAACGCAGAGTATTACATTGTAACAAGCAGTCCATTGTAA CAAGTTCCCTAGGTGATTTGGGGTTCATTAGAGTTTGAGAAACTCTGGTCTAGGCAACTA TGTAATAATAAGAGCTTCCTGTGTCAGAAAATGGCCTAGATATAATGTGGAAGCCTCTGA AGCTCAATTGCAGGTATAAGCGTGCTCATCCATTGAGCTAACTCAGATCTGCTGAACCAG CTTACTGGTAAGCTTCCCAAATAAGGGAGGAAATGAGCACCCAATACCATTCAGATTTGT TTATCCTCAAGAGGCAAACAAACGAAGTCCCTGACCTCCAGGAGCCTGAAGTCTAGTAGG AAAGACAGCTGGAGTGATTATAGACCAGAAAGTATAGATCAAGATAGATAGAACAATTAA GTGGTCTACTCAGGGAAGCAGTGGGTGACATGGGAGCATGTAGGATGGGAACCTAACCCA ATCCTGCTGGAACGGGACAGTTTCCTATAGAAAATATCAGCTAAGCTGAAATCTTAAAGC TGAAGAGCTGAAAGAGCAGAGGATGAGATAGAGGGCCTTCTTCAGCCAGAAACCAATTTG GCAATTGGAAGTGAGACTGTTTTACTCTTTTGCATTCTTTTTTTCACCCCAGGACTTTGG AGCTGTGGGCTTACCCTAGTCTCCAACAGCAAGGATGATTCCCTTCCTGCCTTCAATGTT GACGTAAATTTCATGGGCAGCTTTAGTAACAGGCCGTCTTCCTTTCTTTCATTCTCAGTA ACTCTGTAAAGGCCAAAAGGACAGCTTATACGCTAGTTATATAGACCCTGTAGAATCACA TACAGTTTGCGTGAGGGAAGTGTTTGGATGTAGCATTAAAAAGGGTCTCTCTGAAAGCCA GGATCTCTTTTTCATAATTACAAATAACAATGTTTAATATTCAAAGTAATTTACACTGGG AGCTTTAGCCTAGAGTGTTCCAAATTTTGTTAATGTCATGGCACACATAAGAAATGACAC TATTTGTAGGGAATGGTAAGGTAAATGGATAAGGCTGTTTGTAGGGATAGATGAGGGGCA TCAGGGTTGGCAGGATTTAGAGAACGGGCTAAGGCACTTGGACTTAATGTTATAAGCAAT AAAGAGACACTGAATTAATCCACCAAGGCAATTGGGAGGATGAGAGATGGTCTATGCCAC TGGGTGGTCCTGGGAATGTTGGCAAGACATATTTGGCAGGAGTTGAAAAGATGAGGAAGC CCAGGAAATAATGTGCTGATCCATTAGGAAGATGTTTTAATATTCAGGGGACCAGTAATA AGCAGTTGAGGAACTATTGTGACAACAGAAGAAAGAAGATTTTCCTTCTTCCATTTTTAC TTACTCAAGGAATGGATTCCATTGGAAAAGTTTAGCCTTAATATCTTTTGAGCAGAACCT CATTACGAAGAGAGATACCAGTTTTTATTCATATAAGGAAGAAAAACAAAAAGTAAAGTT ACCAAGGAGTTCTTTGCAAATATTCAACCTGGCCTATGGCTATGCTTTTTCATTTCAGAT ATTTGAAGATGTTCACCTTGAGAACCAACCCAAAATCCAAGAATTACTAAATTGTACTGA CATTATTTTTACACATATTTTTATCCTGGAGATGGTACTAAAATGGGTAGCCTTCGGATT TGGAAAGTATTTCACCAGTGCCTGGTGCTGCCTTGATTTCATCATTGTGATTGTAAGTTT ACCATCTCTGTGTCCCACACAGGCTGGGGTAGTGGTTAAGGCAGGGATCATATGGAGGCT GCTTTTCCTGTGTGCCGCATATGGATGTCTGTCTAAAATAGTTTTTCTTGTTATCATTGC CTCTTCACAGTACTTGTAAGTAGTTGTCATTTGTCTGTAAGCAGTTTAGCCTTTCCTAGG TCCCGCTCATCATACACTACACTTTATATGCTCACCACTACAACCTTTCCCATGATGGGA TTCATTCACTTCTCTCCATAAGCACTGTTTATGTATAATATGTACCTAGAATAACTTTCA GTATCCTCGATTTTTTCTTCTTCCAACTTTCTATTTCTTTTCAAGACTTCTCAAAGCTGG TCAGCAGCCAATAGACTAGGCTAATATCCATCTTAATTGTCAAGCAGCATAAGAGGATTG TTCACATACAGTCTCTCATTTAATATGCATGACAATGTTCTGAATTAAGCAGCATTGTAT GACACCTCCCACACTGTATACAAGGAAACCTGAAGCTCAGAGGATTATCAATTTGTTTCA GGTCATAGAGCTGATAAGTGGTGAAATTGGGATTCACGTGTCTAGCTGTATGACACAAAA GCTAATAATAGTATTAGTGGATCACATTTATTGAGTGCTTTCTCTGTATCAAGTACTCTT CCATATGCTCCAAATGCCTTCTCTCTTTTAGTTGTCATAGCCACTCTGTGGCAGGTGCTA CTATTTTTGTATATTCAGAGAAAGAAAGTAAGTCATAGAGAGGCTAAGAAACATGTTCAG GATTGCCCAGCTAGAAGTGGTGGAGGTGAGATACAAACCTCGTAAGCCTCACTCCAAAGC TCTTGATTTAACATTACATCCTTTTTTTAAGATTAATTTTATGTTTACTGATACATAGTA GATGGACATATTTTCAGGATACATGTGATTATGTAATACCTTTATATAATTAAAACTGGT GCAACCTAGTAAGTCTCACTCCAGGGCCCTTGATTTTAACATTACCTCTTTCCATCATGA TGTAATTAGTTAAACTTTGTCCCTGCTATGCAAGGAAGTAGATAAGACAAATATCTGAGC ATAAACCATGTGGTCAGGAAACCCCACTGGAGAACCATAATGTTGTTGGTTAGCACAAAT AGACCATGTGTTCATTTAATATGGACATCAATATTCTGAGTTAATCAGTATTACATGACA TCTACCACATTACTAACAAGAAAACCAGAGGCTCAGAGACAAGGAAACAGAAGCTCATTG TGGTTCAGGCATTTTAGAAAGGTATAAATGCCATAGAAACTACAACAGGATTCAAAGATG GGAATAAAAACATCCACGTTTGTCAGTCCTATTAATTTCTAGAGAGCAAATAACTCTGTA CGTATTATCTTAATAGATTATAGTAAAAAGAAAACCCTAAAAACTGTGTGAACAGATGAT TGTTATCTCCATTATAGGGAACTCTGTTTTACCGGATACAAGCCCTACATTTAGGAGCCT GGGTCCTAAAGCTAATTGGTCTACCCTCTTTTTGTGTAACCTTAAGCAAAACATTTCCCC TCTTTTGACAATAATTTTCTCACAGGTAAAAATGAGGATGTGGACTATAAATTGCCCAAA TTCACTGAATACATAATAGAAAGCTAGTGATAGTTTCACTAGAAATCTAGTGATCGCCAT TAAAAATATCAAAACAGTTGTCCAAAGCCACACTGTCAAAAAAGGGTACCAGGCTGAGAT GGGTGGGTTTATTTATTTATTTATTTATTTATTATATTTCAATAGGCTTTCAGGGAACAG GTGGTGTTTGGTTACCTGAATAAGTTATTTAGCGGTGATTTCTGAGATTTTGGTGCCCCA TCGCCCGATCAGTGTACACTGTACCTAATGTAGTCTTTTATCCCTCACACCCTCATTTCC CCTGAGTCCCCAGAGTCCATTGTATTATTCTTACAACTTTGTGTCCTCTTAGCTTAGGTC TCTCTTATGAGTAAGAACATAAAATGTTTGGTTTTCCATTCCTGAGTTACTTCACTTAGA ATAATAGTCTCCAATTCCATCCAGGTTGCTGTGAAAGCCACTATTTTGGTCCTTTTTATG GCTGAGTAGTATTCCATGGTGTATGTATATAGTATATACCAAAGACTATATATATATGGT ATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATA GTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATA TAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATA TATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATA TATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCA TATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATAC CATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATAT ACCATATATATAGTATATACACCATATATATACCATATATAGTATATACACCATATATAT ACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATAT ATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATAT ATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCAT ATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACC ATATATATAGTATATACACCATATATATACCATATATATACACCATATGTCTCTTTCTCT CTCTATATATATATCACATTTTCTTTATCCACTGGTTGATTGATGGGCATTTGGGCTGAA TCCATATTTTTGCAATTGCGAATTGTGCTGCTATAAACGTGTGTGCAAGTGTCTTTTTCA TATAATGATTTCCTTTCTTCTGAGTAGATACACATTAGTGGGATTGCTGGATCAAATGAC AGATCTACTTTTAGTTCTTTAAGGAATCTCCACATTGTTTTCCATAGTGGTTGTACTAGT TTACATTCCCACCAGCAGTGTAGAAGTGTTCTCTTTTCACCACATCCACGCCAACATCTA TCATTTTTTGATTAGTGCCATTGTTGCAGGAGTAAGGTGGTATCACATTGTGGTTATGAT TTGTATTTCCCTGATAATTAGTGATGTTGAACATTTTTTCCTATGTTTGTTGGCCATTTG TAAATCTTCTTTTGAGAATTGTCCATTCATGTCCTTTGCCCACTTTTGGATAGAATTATT TGTTTTTTTACTTGCTGATTTGATTGAGTTCTTTGTAGATTCTGGATATTAGTCCTTAGT CAGATGTATAGATTGCAAAGATTTTCACCCACTCTGTGGATGTCTGTTTACTGTGCTGAT TGTTTCTTTTGCTGTGCAGAAGCTTTTTAGTTTTAAGTTCCATCTATTCATCTTTGTTTT TGTTGCATTTGCTTTTGGGTTCTTGGTCATGAAGTCTTTGCCTAAGCCAATGTCTAGAAG AGTTTTTCCCATGTTATCTTCTAGAATTTGTATGGTTTCAGGTCTTAGACTTAAATCCCT GATCCCTCTTGAGTTGATTTTCATATAAGATGAGAGATGAGGATCCAGTTTCATTCTCTT ACCTGTAGCTATCCAGTTATCCCAGCACCATTTGTTGAATAGGGTGCTCTTTTCCCAATT TATGTTTTTGTTTGCTTTGTCGAAGATCGGTTGGCTGTAGGTATTTGGCTTTATTTCTGG GTTCTTTATTCTGTTCAATTGGTCTGTGTGCCTATTTTTATACCAGTACCATGCTGTTTT GGTGCCTATGGCCTTATAGTACAGTTAGAGGTCTGAAAATGTGATGTCTCCAGATTTGTT CTTTTTGGTTAGTCTTGCTTTGGGTATGCAGGCTCTTTTTTGGTTCCATGTAAATTTTAA GATTGTTTTTTCTAGTTCTGTGAAGAATGATAGTGGTATTTTGATGGGAATTGCATTGAA TTTGTAGATCGCTTTTGGTAGTAGGGTCATTTTCACAATGTTGATTCTACCCATCCATGA ACATGAGATGTGTTTTCATTTGTTTGTGTCATTGATGATTTCTTTCAGCAGTGTTTTGTA GTTTTCTTTGTAGAAGTCTTTCACTTCCTTGGTTAGGTATATTCCTAAGTATTTTATTTT ATTTTATTTTCAGCTAGTGTAAAAGGGGTTGAGTTCTTGACTTGTTTCTCAGCTTGGTCA CTGTTGGTGTATCACAGTGCTACTGATTTGTGTACATTGATTTTGTATCCTGAAACTTTG CTGAATTCATTTGCCAGTTCTAGGAGTTTTTTGGATGACTCTAGGATTTTCTATGTATAC AATCATATCATCAAGAGGCCCAGATGGTTTTGCAGGTAAGTTTTACCAAACTTCCAAGGA AGAATTAATCTCATCTTATTCAAATTGTTACAAAGAATAGACAAAAGAAGGAAAGCTTGC AAATTTATTTAATGAGATAAACATAATATTGATAATAAATGAATAGAACAGAAATGAATC AAATAAGAAAATAATCTTACAATTATATATCTTTGCAAATCACAGATAAAGCACTCAACA AATGTGGTTTACTATTACTATTATTAGCCTTGACATCATGCAGACTGTGGTGTGAATCTG AAATTCACCACTTATCAGCTGTATGACCTGAAACAAGTTGCTGATCCTTTGAGCTTCAGG TTTCCTCATCTATAATATGGTAGATGTTGCAAAGTGCTGCTTAGCTTGGGATATTGTTGT ACAGATTAAATGAGAGAATGTATGTGAACAGTTCTATAATATTTATTGTCAATCAAGATT ATATAAAATTACCATCATAATTTTTATTAGAAAAGCATAAACCACAAAAAGATACACAAT TCAACTACATCAAAATGTAGTAAAACTTTTATTTTTGTATTCAACAATACTCTAATTACA GTTACAAACCAACTAGAACCTGGGAGACAGTATTTTCCACAACAGATCATTATTATTCAC AACATATACAAATTTCCTACAGATTCACAGGAAAAAACAACCCAGAGTAAGAATGGGAGA AGTATACAAACAGCCAATTCACAAAAGGAATAAACGTATGATGAAATATACACTAGTTCT ATCACCAGTTAGCACAGAAGTGCATATTAAAATGAGAGGCCTCTTTTTACTCATTAGATT GGAAAACAAAATATATTGTTTTGAAACATGGTCTCACTCTGTCATCCAGGCTTGAGTGCA GTGGCAACCTCTGCCTCCCAAGCTCAATTGATCCTTTTGCCTCAGACTCCTAAGTAGCTG GGACTACAGGCGCCCACCACCATGCCCAGCTAATTTTTGTATTTTTTATGGAGACAGGGT TTTACCATGTTGCCCAGGCTGGTCTCAGACTCCTGAGCTCAAGCTATCTGCCTGCCTCAG CCTCCCAAAGTGCTGGGATTACAGGCTTGAGCGACTGTGCCCAGCCATGGCAAAAATTTT AAAGTTGGATAACATCGATATTTAGAAGGATATAGGAAAATGCCATGGCAAAAATTTTAA AGTTGGATAACATCGATATTTAGAAGGATATAGGAAAATGCGATCTCAAACCACTGCTAA TAATTTAATTGGTACAGCCACTGTAGGGAATATTTCGCACTATATTAAGTAAAAATCACA AATGACCCAACTCACAATCCACAAATATCACTTGTTATAGATCCAAGGAGAACTCTTGCA TATGTACCCAAGAAAAATGCACACATCTGCCCAGTGTACTGTTGTGCATAATACTGTCCA GTGTACTGTTTTGCATAATAGGAAAGAAATGGAAATAACCTAGGTATTCACCAAGGAGGA ATGGATCCGTAAAACATGAAGATTTATATGCTGTACTATTCCTCAACAGTTACAAGGAGC AAACCATAAGAATATAATCAACATGATGATTTCAAAAACAGTGCAGAGTGATAAATGCAA GTTAGATTTTAAAAATAAGATAAAACTAGTAGAAGACTTCTCACTTGAAGAGTACAGTAT GGTGGGAAAGAATGTGGGTGTTTCTATGTGTCTCACTGATACCTGCAAGTCTGATGGAAC AGAAACTTGGGTAAGGTTTAAGGTAAAGAGGTGTATAAGGTCTCTGTAGGAACATCCCTT CTCACCAACTATGACAATGATTTGGTAAAACAGAAGAATTGCTAAACTTTGCCATCTGTT TTCATCTGGCTAGGAATAAGTGGAGTATTTGCGAGAAAGCTACTTAATAACTTAATAACT TAAATAACTCTCCAAAAGTGGTGCCATTGTAAACAAAAAGCCCACTAAAAACATTATAAA TACACTGTAAATTGATTATAAATTTCAAAATCTTAATATTCTGTCATATTAACTCAATTT CTCTTCACTACTACAATGAGGTTGGCAAGATTTGTGAGGAATGTTTTTAGGTAATAGTGA ACTCTGACATCAGCTTAGTCAGGACTATCCTGATTCACTTCCTATCCAGCTGAGCAACTC TTCTTCCAAGCTGAGCCTCTGAAAACTCTCCATAATAAGAATGGCTGAAGATCTGCAGCA AGCAGATTCAACTCAAGCTCAGCATTGATTCCATTGTGATAATAGAAGCAAATCCTAATT CCTCATATGAAAGCCAATACCTTCTCGAATTGTCCTCTACTGAGCATCTGTAAATATTGT TAAGATTTAATTTGGTATACTTAACAGAGCTTTCTAATACTCAGTTCTGTCCTATTGAGA AGAAGCATATTGGGAGAGAAAATAGCATTTAATTCCCCAATAAGGAGCTATTTGGATTAG GGGAAATATTAGCAAATAATTTATTTTTTCCATGTAACTTTTTATTTTGATATCATTTCA AACTAAGAAAAATGTTGCAGGAAATAGTATAATGAAATCCACATATTCTTTCTTCAGGTT CACCAATTATTGTCACCCATCTGCTTTGTCATTTTTTTCTCTCTTTTTTTCCCCCTAAAC TGCTTGAGATTAAGTTGCAAGCATCAAACCTATTACCCCTAAACATGTCAATGTATATTT CCTATCAAGGACAATGTCTCACATGATTACAATACAGTGATCAAAGTTAGAAATTTAACA TTGATTCAATTAAAATCCACATCCATTTTCAAATTTTTCAAATTCACCATAAAGATCTTT GTTATGGCAATTGACTATCATAATAAATTTCAATTGACTGCCATAACAAGATTCTTTATG GTTAATTCCCTTTATGCATCCACCTCCTACCCCCACTCCAATCCAGGATCCAATTCAGCA CCCCAGATTACATGTGATTGTTATATCTCTTTGCTTTCCTTTAATTCACAACAATTTCTC GGATTTTCTATGTCTTTCACTAAACAAATAACGTGAGTGCATTGTTCCCACGTTTATGGA CTGTAGCTTTGGTAGATAAGGAGAAAAATGGACTTTCCAGCTGGACACCAAGTCAGGTAC ATTATCTGTGGCCTCTCTTCCCATCAGGTCTCTGTGACCACCCTCATTAACTTAATGGAA TTGAAGTCCTTCCGGACTCTACGAGCACTGAGGCCTCTTCGTGCGCTGTCCCAGTTTGAA GGAATGAAGGTACATTCTGCAGAAGAATGGGTAGAAGTTCAGTTAACAGAGAAAGGTGGA AAGACCAACAGTTCTTTTTGGGCTGAGATTTCCTTAAATTGCCAAGCTTTTCCTGGGTTA CTTCCCAGCCTGCCCAGTGCTTAGAATTTGAGGGGTAGAGAAAAGCCTAAGATATACTTT CTACCCTAAAAGCTTCTGTGACAGCCAAGATGAGCTGTAGCTTTGCCTCAGAATCCTGGG GTAGGTGTCCATAAGTCTCTAACACTGTCATCACCAGGGCACTTCTCAAGGTATCCTCAG TGGCAACAGAGAGGACCCATTATGTAGGGAGCTGAGAGCAGGGCTTGTCATGGGGATGGG GCACATGTATGTGGAGATTAAGGACAGAGCTCCAAGAGATCAAGCCCTGCACATCAGGCC CATGCTGAGAAGTTGAAGAGCCTGGTGCATCCTACCCATCTGTTATGGTTTTCTTTGCTT TTGTTTCCATAAGGTGGTGGTCAATGCTCTCATAGGTGCCATACCTGCCATTCTGAATGT TTTGCTTGTCTGCCTCATTTTCTGGCTCGTATTTTGTATTCTGGGAGTATACTTCTTTTC TGGAAAATTTGGGAAATGCATTAATGGAACAGACTCAGTTATAAATTATACCATCATTAC AAATAAAAGTCAATGTGAAAGTGGCAATTTCTCTTGGATCAACCAGAAAGTCAACTTTGA CAATGTGGGAAATGCTTACCTCGCTCTGCTGCAAGTGGTAAGTACAGTCAGTATGTTTTT CACTCTGAGGAGAAAAACCTTCAGAGAATCATGAATTGTTTCTCAGCTAATAAAAATGCA GTGACTCCATTGTGATAATAGAAGCAAATCCTAATTCCTTATATGAAAGCCAATGCCTTC TGGAATTGTCCTCTACTGAGCATCTGCAACTTCATTGACTGCTGAGCCTCAGCTTCCTAG TGATGCCCCTTTAAGGATCTGTACATCTCACCTTGTCCTGTATGGCACCTTGCTGGCTCT CCACATCTCCAGGCAGAGAGGAGTGGAGCCTCAGGTGTAGGTCATGACACTTTGAACAGT TCCCTGTTTAAAGCACAGTTGCCAGGTCTTGAACAGGCAGTGCAAATACATATGGCATCA GAAAAGAACCCTTTAACTCTTAGCCAGTCTCTTTTCAGCTGTTGGGCCACAGCTTCAGCC TCTCTGGTAACTTTTAAGGTGGAAATTTCTTGTTTTTAAACTTTGTATCTTGTACAAATG AAGAAGAAGAAAAAAATCCAAGACACACACCCTTGAGTTCCCTTCTACTGTTCTTTTATT TGAGCATCCTGTCCTAGAAGGTTGGGGTATCATCTTATATATTCAGCCCACTGGGCCTCA CCTGCTTTTCCATGCCATGTATCTGTTGATGGCAAGTCTACATTTGTATTATTCATGTTT TTCTGCTTTTGTTGCAGGCAACATTTAAGGGCTGGATGGATATTATATATGCAGCTGTTG ATTCCACAGAGGTGAGTCAGTGTTCTACCATGTTCTGGAGTGTTATGGTCAAGTCAGAGA TATCATGACTACATGGACAGTCCAGAACTGGCGTCATAGTTCCAGCAGCTGGGGTTCTCT GCCTTGTTTTCTTTGGAACAAAACACTATGAGATACCACTGCCTAATACAAAGGATTCTT ATGCATCCTCTAGTGAGTATCTGATACCCCAGTATTCTCTGAAGTCCAGTCTTCTTTCTC CATGGTGGCCAAGGAAAAACCACTCATACAGGAGATGGAGCCAGAAGTGGCAGAATCACA GGATGCCTTGATTTTCTTCTTCCCTCTTCTCCAAATGGTTGGATAGTCAGAAACCAAATT TCAAAGAGTATCTGTTTTAGATTGTGGGAAACCTTAAGACTGCACTAAATTTGCAAGGGA GACCCTCGCATGGCTCCAGACTCCCTAGTGATGGCCAACTGTTGAATTTTAGAGTTTCTA GTGATGACCAGCTGTTGAATTTTAGTTAAAGAAAAGATAATCACAGATTCCCAAAGTTGG AATTGCACTCAGTAATCTAGCCTAGTTTCTATTTTCCAGCTTGGAAAGTTGAGGGATAAA AAGAAAGAGTTTCCCAAAGCCATAGAGTAAGCAGATGGTTGGCTCAAGAATGGAGTCAAT GGCAGAAATTATTGTTTCCTATTATAGCACATGTGTGTGTTTCGACCTATTTGGGAGTCT GCAGAGTTGATGCCTAACACCTAAAAAGTATTCAGTACCTGTGAGCTGGTACAGACTGAG TATCCCTTATCTGAAATGCTTGGGACCAGAAGTGTTTCGGATTTTGGAGTTTGTAAGATT TGGGAATATTTGCATAATACTTACAGTTGAGCATCCGTGATCTGAAAATCCAAAACGCCA CAGTGAGCATTTCCTTTAAGCATGACCTTTGCATGTTATGTCAGCATTCAAAAAGTTTCA GATTTTGGAGCACTTGAGACTTCGGATTTTCAGATTAGGGATACTCAGCCTGTAGTAGCA GTAGAAGTGGTTTGATTAGTGTTATAGCAGCACCGAGGGATAAGAATTAATCTTTATGGC CATTCCAGTTGTCATTATAAATTCACAGATTGTCATTATAAGTTAATATACAGATGATGC ATAGCAACAAATCTGAGTTTGCTTAATTCTGGGTGTGTCAATAAAGTCAACTTTCTGGAA AGACAAAGGTAACAGTAGATACTTAACTGTTTATAACAATTATAGTTTACATTGTAATAT TTATATATACAATGTAATTTTATAATTATATATACAACATAATTTTATAATTACAATTTA TAGCAAAGGAAATATTTTAAAAATACTAATGTACTCATTTCTTTCGTCTTTTCGAGTACA TGACCTTCACTCTAGTTTGGCTTTGACATCCATCTGTGCGGAGGTGGAGATGGGGAGTGG GGAGGAAAGGTTTTGCAGATGTGTAGTAGACAAGGTGTGTGGCTTGCAAATGCTCCAAGA GTGAGTGACAGGTGGGTGGAAGTGTGTGGAGGGGTGCCTGTTTTGATGCCGGTCCTGATA TGTACTGGTTGTTGTATGCTGGCTTCTAGCTTGATGACTCTTGTCTATATAGTCACCCAG CACTCTGCTGGCCTCAAATATTGAAGTTGTGGTTGTTGAAACTGTGAGCGTTTTCCCCGT CATCTCCTCCTGGCCTTAAATACTGGTACTCTGCCATATCCTCCCCATGGTCCCTGGCAG TCTACTCCCATCCCTTGCTGCAAGGCCCAACATGACAGGCCCATGGCTATCAACTCCGTG TCCAAGCCACATGAGAACTGAGAGGTTCAAGAGACTTCTCTCTGTTCAATTATGTTACTC AAATCTCTTAAGTCAGCCTGCCTTCCTGATATATCTCAGGCCTTCCCAGTAGTAATTCCT TCCAGGAAATGCAGAAGTAAAGCAAGAATACCCTCTTTGCCTTCCATGTTTCCCTCAATT TACTGCATTCCAACCCCCCTTTCCAGACCCAAAGAGGGATAATCAGGACACACATCTTGC AGCTCTGATTTTCTGTCTCTGTTTATTTTTCCTACATTTCCTATGAACAAATTGGGTGAG CTTTGATAATTTCATCTTCTTGTCTAAATCTCCCTCCAATAAAACTCTCCACCTTCAAAC CTTTGATTTAACAAAGAGGGACATGAAGGAGTACAGACATTCCTATATCTCAAAGCAGTA ACCTAACATGCAAGACTATTAGCCCACTTGGCAATTTTATTTTAAATGTCTAATGCTAAA TATTCTTTCCATTACGTTGTTTCTGTACTAATGTCTACCTTTTGTTCAAATAAATGGATG CCTCAAGCAAACTTGGACTAAAGTCAGGTAGTCTTCATAGCAAAAGAGAGAAATAATGTT TTCAAATAATTACCTTCATTACACTAGTCTACATTGTGAGTGTTGAATAACAAATACCAT AAATGCATTAAACATTAGTCAGAGCTATCAGCACTTATATCTTTGATGTGATAGAGGGAA AAAAAGTGAAATCTTTTTGCCACCTGCCTGAAAGTGCTTGAAGCCAATAACAATGCAAAG GACTTGGATGAAAGGGCTCTTAGGTGATTTGGGTTGTATCTTAGACTAGGAGAGCTTGGT TGGAGTCCATTTGTGTCCATTTTAGAGCTCGATCATTTATTCATTCATTTAGATATTTAT TCCATGTGTATCAAAAATCTACATATCAGAAGGTATGCTAGGCACTAGGAAGTTAAGAGT GAAAGGACATTACCACATATTAAGTAAAGCAGTGGGCTCTATATGGGGGTCATAGTAGTT CAACAACACTTAGTAGTTTAGTCACCTCTGCCCAAGGTGAACGTGCCCCTCCTCTGGTGG CCTCACCTCCAGTCAGGTTCCCTCCAGTTACATACCCATATGGACATTCCATATGGATGA CTTTTTAAAAAGACAAATGTGATCATACCACTCATCTCCCTTACAGTCCCTCAATGATTC TCCTTTGCTTAGAATAAAAAGCCCGATTTTCCATCACCCCAGGTCCCCGCCACATGCCAT CACTCAAGCTGAGCTGCAAAAACTGAAAGACAGGCTCCCAACAGGGGCTATGGCTGTTAG GAAGAGGCTATGTAGTCAATGTTGCTGCTAAGAAACACCTTGGTCTTCTAGATAAGGTAG TTAGAATGCTTATATTTTTCTCCAGTAATTGTTTTTTTCTCTTATTAAAAAAATTTCTAA CAGAAAGAACAACAGCCAGAGTTTGAGAGCAATTCACTCGGTTACATTTACTTCGTAGTC TTTATCATCTTTGGCTCATTCTTCACTCTGAATCTCTTCATTGGCGTTATCATTGACAAC TTCAACCAACAGCAGAAAAAGATAAGTATCTGGGTTGTCTTGATTTGGTAATTTTATCTC TGTCCTCCAAATGCATTAACAAAACATAATTCTGGCCTAGAATGTTATACAAAGTAGCCT TTATGATCTGCACAATAACAAGGAGGGAGCCTCCCATGTCTGCCTGGAGATGATCCTCAA AGATGAGGCTGAAAGAAGACTGAAAACATCTGGGTTTAATTAAGTCATTACTTTTATTCT CTTTTACCTTCTTAATCAATGGACATTGCTACTATTGAGATGAAACCCATTAATAACTGA ACCCTAATAGAACAACAACCACCAGGATTAGTATTTATTAAATATGACACTAGGGTCATA TTTTAATAACCTTTCAAATGCACTTTATTATCCACTATAAAACTAATATACTCTCCTTTT CCTGCCAGTGTGTCCTGGCCCCCAGCACCCAAGCCTCAGCAGATCTATCCACCCTACTCG GAAAGAAGATGATTGGGGAAGAGGCAGGGCTAGGGAGGTTTTCTTCCCTATGCATTGTAA TAAACACATGTTTATTTTGCTGATTCTAAAATCCACAGGAGCTTTGTAGTGAATGTGGAA AATCAAGATAAAAAGAAAATTTAAATCACCCAAAGGTGGCCACCAAATGATAACCACTTT AGACCATGCCTCTTATGATTTTGATATAATCCATTCCAGTCACTTCGTATTACTTTTACA CAGTTAGGATTACCTTATATTTTTAATTTTGTAGTTTTGACCTCATTAGTTATAAGCTTA TGTCATAAAATTAACTTTGCTATAAGAATTCTCAGAGCCTCTGGCAGGGCGTGGTGGCTC ATGCGTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGGAAATCACTTGAGGTCAGGAGT TCGAGACCAGCCTGGCCAACATGGTGAAACCCAGTCTCTACTAAAAATACAAAAAATTAA CCAGCTGTGGTGGTACACGCCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATT GCTTGAACCTGGGAGGCAGAGGTTGCAGTAAGCCGAGATCGCTCCACTGCATTCCAGCCT GGGCAACAGAGCGAGACTCCATCTCAAAGAAGGAAAAAGAGAAAAAAAAAAAAAAGGAAT TCTCAGGGCTGAATTACATTGCATATTATGGATAGACCATAATTTATCCTGATCATTCCC CTCTTTGGACATTTATAGGTTGTTTTTAAATTTCTTGCTCTTCTAAAACAATTATGACCA TTTTTAAAATTCTGTGTATCTCTGATTATTTCTTCAGCATAAATTCCTAGAATTACAATA TTAAAAGCATTGAAGACATATTGTCAAAATACCTTCCAGAAATGCTGTGCTAGGTACCCT TATTCTGCAGAATATGAAAATGCATGGTTACTGAGATCTCATCATTCTAATTGTTATATA TTTATTATCTTTTTTAGACTGGTGGGGAAAGTATTATCTCATTGTTTTATATTTATTTAT TTGATCATTAGTGAGGTTACACACTTTTTCACAGGTTGATGGCCACAAGTATTTCTCCTT TAAGAGTTTTTTATTTATATCATTTGTCTATTTTTAAGTCTGAGATTGCATTTTTATATA AGTCCAATTTGTGACATGCATCTTATATTTTTGTAATAACATATTTAACTTATAGACATT AGTATTTTTCAGAGTCAAACTCCCAATCTTGTTATTTTTGGCTTTTTTTTAATTGTACTT ATGAACTAAAGGCATTTCTCAATCCCCAAATCAAGTAACTAAATATTCTCCTTTGGTTTT CCTAATTGTTTTATGGCTTTCTTTTATTTTTCCTCTGGACTCTCATAATTCATCCAAAAT GTACCTTGATACCAATTAGCCTTCTTAGCATCTGCTGGGAAAGTTGTTCCCTGTTGTTAC ACAAACTGTATTGCTTCTGCTACTCAGGAGTGTGTGATGTAGCATCCTCTCTGTAATATG CATCTTGATTGCTATCAATACCTGCAATTTGGATGTCAGTCTTTGTGATTTTAGATTTAA GCTTACATTAGAAATCATCGTTTTGAACAATCCACTATATCCTTTTGTTTTGTCTTGTTT TACTTTTTATTACAGAAAATCTCAAACATGCATAAGAGTGTACTAGAATTAACCTAAAAT ACCCATCACCTAGCACAACATTTATTGACTTAAGAATGTGCTTGTTCTAAGCCATATACG CCCTTACTCAACTCTGCCACCAGATTTTTTTTTATGGAGCAAACCACAGACATCACATCA TTTGATTCATAAATATTTTAGAACACATCTCTTTTTAAACATAACCACAATTCCATTAGT ACACCAAAAAGTTTAATGTGAGTTCTTTAAATTCATCAAATACCGAGTCTATTTTCAAAT TGGTCTGAGCCTTTAATTTAAAAGAAAAATGTTTATTTATTAATTCTGTGTTTCATTTGC TTATTTGTTTCTGTTGAAGCTTTTTGTTTGTTTGTTTTCAAATCAGGATTCAAATACATA AGGTCTTTCCATTTGACAACTTTCTTGTTCTCTGAAATGCCACGATGTTCCAAGTTTAGG TTGTACCCTTCCTGCCCTAGTTCTGTAATCAGCTATTTCTCTAAAAACTGTTTGTTTCTT TGTGGGAAACGGTTTCCAAGACCACAGTCTAGAATGCTCATTGTCATTGTTTCTAAGCCT TTCTAGTGGACAGAGTTGCTTAAAATTCAGGACTACAAGGAAATATATAAGTGGAAATTT GCATACTGATTACTCAAATTCAGGACTACAAAGTTTTCACTTAACATCTATATCTCCCCC TTTCCACACTGAGAAACCTTATTCTCAAAGGCAAAGATGATAGAATTTAAAATTTCCACA ATTACTCATTTTCTTTATCTCACAACACACATACACACACTACAGTCTCAGAATAGTGGT ATTAATACTACTATGACATATATGATTACTAAAAGACTAATTTCTTCCCAGCATCCCAGA ACAACTTGATTAATTTTTAGGTTGACTTCTAGGGCTACATATTTTTATCTGTGCTGGTGG AGTAAAGTCAAACCCCAAACCCTCATCAAAGTAAGCCTGAAGTCACAAATGATCAGGGAA GACTGGCTTTTTACTCATAGTCTGGATTGAGACAATTTTCTGGTTGTCTTCCATTACCAA CAGACAGATTCGGGTTTTTTTCTTTACCCTTCTGTTGGCCACCTTTGGATAGTCCCATAT TCTTGCAAAGACATCTGTGCCTCTCCTGTCTTTGCGTAGGCCTTATTTCCCATTACCCCC TTACATTTACTAAAAACTGAGATTCTAGTTTACAGAGATTAGTAAATGTTCTAAGACCAG CCATGGCTTTGGGCTAATTTACTAGTCTATCTTTTTGATCTCTCAATTTTCAGACCCCCA ATTTTTTTCTTACTTTTATGCCAGTTCAGCTTTACATTTTAAAAGAATTCTGTTCTAGTT TATCTAGCCTTTCTAAATATTTTGCCCCCAAATTTCCAGATATGAGAATCCTAGTTCATT TTTCTATTCTTTCTTGCTCAGTAGTGAAAATAGTTAGAACTTTTTTTTTTCCCTGAGAGT GGCTCTGAACAGATTTTATGCAGCTTTGTTAGTAGTGTTATGTGTATTCATTATTTAAGA GAAATTCAATTGCCCTTTATGAGATGTTTGTCTAAATTTTTGTACTAATTTCTATTTTTG TTGCATCATGGTTTGATTTATGCAAGCTGAAATATATCAAATTTTTGTAAAGATTCTGTG TATATTTGCGAAGATTTGTAACCTTTCTGTTCTAAAAAGTGAAAAATAACTGATAAAGGT AACTTTATTTCTCCTGTATACCTTAACTTAAATCTTGTTTCTTTACAGCATTTCGCTATG GTCCAGTCTCTTAAGCTTACCATTGAAATTTTAAGAATTGTTAATAGAAATGTAATGTAA CTTTAAAATTGTGTCAAAAAATTCAGAACTAGATTAAAAAAAAAACTGTTAAAGGTAATT AGCTCTTATCTTCTCCATATACTTAGGTGGCCAAGACATTTTTATGACAGAAGAACAGAA GAAATACTATAATGCAATGAAAAAATTAGGATCCAAAAAACCTCAAAAACCCATTCCACG GCCTCTGGTGAGAGCAATTGAATGACTGTAAATATGTAGAACAGTTGAGTTACTGTGAGG AACTCAAATTTCCGTAATGCCATCCTTCTTTATTCTTAAAATTTGCAGACATTAAGGACT CAACAGAAGTCAAGACATATTCAAACACTTAGGAAAATTAGATACTCAAACTCAAAAGTA AGAGCCTGGACAACAACTTAAATTTATTTTTGAGTGAGCCAGGCAATATGTGCTTAAGTA AATACTTATTGCGACCATGTGAACTTAGTGGTTTGTTTGAACTTAATTTTGACCAGTTTA TTTTATTTTATTTCAGTAGTTTTTGGGGAACAGGTGGTTTTTGGTTCCATGTGTAAGTTC TTTAGTGGTGATTTCTGAGAGTTTGGTGCACCTGTCACCCAAGCAGTGTACACTGTACCC AGTGTGTAGTCTTGTATTCCTCACCCTCTTCCCACCCATCTCCTTGATTCCCTGAAGTCC ATTATATCATTCTTATGCCTTTATGTCCTCACAGCTTAGGTCCTACTTATAAGTGAGAAC ATACAATACTTGATTTTCCATTCCTGAGTTACTTCACTTAGAATAATGGTCTCCAACTCC ATCCAGGTTGCTGCAAAGGCCATTATTTCATTCCTTTTTATGGCTGACTAGTATTCCATG GTGTAGATATACCACATTTTCTTTATCTGTTCTTTGGTTGGCAGGCATTTAGGTTGGTTT CACATTTTTGCCGTTGCGAATTGTGCTGCTACAAACATGTGGTATGTGTAAGTGTCTTTT TCATATAGTAATTTCTTTTCCTTTGGGTAGGTACCTAGTAGTGAGATTGCTGAATCCAAT AGTAGTTCTACTTTTGGATCTTTAAGGAATTTCCATACTGGTTTTCATAATGGTTATACT AGTTTATATTCCCACCAACAGTGTAAAAGTGTTCCCTTTTCACCACATCCATGCCAACAT CTATTATTTTTTTGATTTTTAAATTATGATAATTCTTGCAGGAGTAAGGTGGCATCCCAT TGTGGTTTTAATTTGTATTTCCCTGATAATTAGTGATGTTGAGCATTTTTTTTCTATGTT TCTTGGCCATTTGTAAATCTTCTTTTGAGAATTGTCTGATTATGTCTTTTGCCTACTTTT GGATAGAATTGTTTTTGTCTTGATGATTTGTTTGAGTTCCTTGTAGATCCTGAAAATTAG TCTTTTGTCAGATGCATAGTTTGTGAATATTTTCTCCCACCTGGTGAGTTGTCTCTTTAC TCTGCTGATTATTTATTTTGCCGTGCAGAAGATTTTTAGTTTAATTAGGTCTCATTTATT TATTTTTGTTTTTGTTGCATTTGCATTTGGGTTCTTGGTCATGAATTCTTTGCCTAAGCC AATATCTGGAAGAGTTTTCTTGATGTTATATTCTATAATTTGTATGGTTTCAGGTCTTAG AGTTAAGTCTGATCCATCTTGAGTTGATTTTTGTACCAGGTGAGAGATGAGGATCTGGCT TCATTCTTCTACATGCAAATTGCCAATTATCCCAACACAATTTGTGGGATAGGGTGTCCT TTCCCCAGTTTATGTTTGTTTGCTTTGCCAAAGATCAATTGGCTGTAACTATTTGGCTTT TTTGGGGGGTTCTCTATTCTGTTCCATTGGTCTACATGCCTGTTTTTATACCAGTACCAT GCTGTTTCAGTAACTATGGCCTTGTAGTATAGTTTAAAGTCAGGTAATGTGATGCCTCCA AATTTGTTCTTTTTGCTTAGTCTTGCTTTGGCTATGCAGGCTCTTTTTTGGTTCCATATG AATTTTAAGATTTTTTTTTCTAGTCCTGTGAAGGATGACGGTGGTATTTTGTTGGGAATT GCACTGAGTCTGTAGATTGCTTTTGACAGTATGGTCATTTTCACAATGTTAATTCTACCT ATCCATGAGCAGGGGATGTGTATCCATTTGTTTGTGTCATTAATGATTTTTTTCAGCAGT GTTTTGTAGTTTTCCCTGTAGAGATCTTTCATCTCCTTGGTTGGGTATATTTCTAAGTAT TTTATTTTTTTCTGCAGCTGTTGTAAAATAAATTGAATTCTTGATTTGTTTCTCAGCTTG GTTGTTGTTGGTGTATAGCAGTGCTACTGTTTTATGTACATTGATTTTGTATCCTGAAAC TTTACTGAATTTATTTATCAGATCTAGGAGCTTTTTGGATGAGTCTTTAGGGTTTTCTAA GTATACAGTCATAGCCTTGGCAAATAGCAATAGTTTGACTTCCTCTTTTCCAATTTGAAT ACTCTTTATTTCTTTCTCTTATCTGATTGCTCTGGCTAGGACTTCTGAGTGCTATGTTGA ATAGAAGTGGTGAAAGTGGGCATCCTTATCTTGTCCCAGTTGTCATAGGGAATGCTTTCA ACCATTACCTGTTCAGTATGATGTTGGCTGTGGGTTTGTCATAAATGGCTTTTACTACCT TGTGCTATGTTCCTTCTATGCCAGTTTTGCTGAGGGTTTTATCATAAAGAGATGCTGAAT TTTGTCAAATGCTTTTTCTGTGTCTGTTGAGATGATCATATGATTTTTGTTTTTAATTCT GTTTATGTGATGTATCACATTTATTGACTTGCGTATGTTAAACCATCCCTGCATTCCTGG TATAAAACCTGCTTGATCATGGTGGATTTTCTTTTTGATATGCTGTTGGATTTAGTTAGC TAGTATTTTTTGAGTATTTTTGCACCTATAGTCATCAGGAATATTGGTCTGTAGTTTTCT TTTTTGTTGTGTCCTTTCCTGGTTTTGGTATTAGGGTGATACTGGCTTCATAGAATGATT TAGAGAGGATTCCCTCTTTCTCTATCTTTTAGTGTTAGTGTAACCACCCTTGCCCCCTGC CTAGTCAGAGCCAATTTATCAAGATGGGGGAATTGCAATGGAGAAAGAGTAATTCACACA GAGCTGGCTGTGTGGGAGATGGGAGTTTTATTATTACTCAAATCAGTCTCCCCGAGCATT CAGGGATCAGAGGTTTTTAAAGATAATTTGGTGGGTAGGGGCTAGGGAAATGGGGAGTGC TAATTGGTCAGGTTTGATATGGAATCATAGGGGGTCGAAGTGAGGTTTTCTTGCTATCTT CTGTTCCTGGGTGGAATGGCAGAACTGGTTGAGGCAGATTATGGGTCTGGGTGGTGTCAG CTGATCCATCCAGTGCAAGGTCTGCAAAATATCTCAAGCACTGATCTTAGGTTTTACAGT ACTGTACTGATGTTATCCCCAGGAGCAATTTGAGGAGGTTCAGACTCTTGGAGCCCAGGG GCTGCATGACCCCTAAACCTTAATTTCTAATCTTGTAGCTAATTTGTTAGTCCTGCAAAG GCAGACTGGTCCCCAGGCAAGAGCAGGGGTCATTTTGGGAAAGATCTATTATCAATTTTG TTTCAAAGTCAAGCCATGAACTGAATTCCTTCCCAAAGTTAGTTCAGCCTATGCCCAGGA ATGAACAAGGACAGCTTAAAGGTTAGAAGCAGATGGAGTCCATTAGGTGTGATTTCTTTC ACTATCATAATTTCCTCAGTTATAATTTTGCAAATGCGGTTCCATAAGTAGGACTGGTAC CAATTCTTCTTTAAATGTCTGATAGAATTCAGCTGTTAATCCATCTGGTCCTGGACTTTT TTTTGTTGGTAATTTTTTTATTACTGTTTCAATCTTGCTACTTTTTGTTGGTCTGTTCAG AGTTTCTATTTCTTCCTGATTTAATCTAGGATGGTTGTATATTTCCAGGAATTTATCCGT TTCCTCTGGATTTTCTAATTTGTGCATGTATGTGTTTGTAGTAGCCTTGAATGATCTTTC GTATTTCTGTGGTATCAGTTGTACTATCTCCTGTATCATTTTTAATTGAGCTTATTTTGA TCTCTCTTCTTTTCTTGGTTAATCTCACTAATGGTCTATCATTTTTATCTTTCCAAATAA CCAGCTTTTTGTTTCATTTACCATTTGTATTTTTTTTGTTTCAGTTTAATTTATTTCTGC TTGATCTTTGTTATTTATTTTCTTCTGCTGGGTTTGGATTTGCCTTTGGCTTGTTTTTCT TTCTCTAATTCCTTGAGATCTGACCTTAGATTGTCTGTGAGTGCTCTTTCAGACATTATG ATGTACGCATTTAATGCTACAAACTTTCCTCTTAGCACCACTTTTGCTGTATCTCAGTGG TTTTCATAAGTTGTGTCACTGTTATCATTCAGTTCAAAGAATTTTTAAATTTCCATCTTG ATTTCATTGTTAACCCAAAGATCATTCAAGAGGAGATTATTTAATTTCCATGTATTTGTA TAGTTTTGAAGGTTCCTTTTGGAGTTAATTTCCAGTTTTTTTCCACTGTGGTCTGAGAGG ATACTTGATATGATTTCGATTTTCTTAAATTTATTATGACTTGTTTTGTGATCTATCATA TGGTCTATCTCCATGTGATATGGAGAATGCTCCATGTGCTGATGAATAGAATGTATATTC TGCAGTTGTTGGGTAGAATGTTCTGTAAATATCTGTTAATTTCATTTGTTCTAGGGTATA GTTTAAGTCCATTGTTTCTTTGTTTAATTTCTGTCTTGATTACCCGTCTAGTGCTGTCAG TGGAATACTGAAGTCTCCCAGTATTATTGTGTTGCCTCATTACTTAGGTCTAATAGTGAT TGTTTTATGAATTTGGGAGCTCCAGTGTTAGGTACACATATATTTAGGACTGTGATGTTT TCCTGTTGGACTAATCATTTTATCATTATATAATGTCCCTCTTTGTCTTTTTTAACTTTT GTTGCTTTAAAGTCTGTTTTGTCTGATGTAAGAATAGCTACTCTTGCTCACTTTTCATTT CCATTTGCATGGAATGTCTTTTTCCACCCTTTACCTTAAGTTTATATGAGTCCTTATGTG TTAGGTGAGTCTCTTGAAGACAGTAGATGCTTGGTTTGTATCCATTCTACAATTCTATGT CTTTTAAATGGAGCGTTTAGGCCATTTACATTCTACGTTAGTATTGAGATGTGAAGTACT GTTCTATTCATCATGTTAGTTGTTGCCTAAATACTTTGTTGGGTTTTTTAAAATTATGTT GTTGTTTTATAGGCCCCGTGAGAATTATGCTTTAAGGAGGTTTTGTTTGTTTGTTTGTTT TGTTTTGTTTTGGTGTATTTCAAGGTTTGTTTCATAATTTAGAACTCCTTTTTGCATTTC TTGCAGTGTTGGTTTGGTGGTGATGAATTCTCTCAGCATTTATTTGTCTGAAAAAGACTT TATCTCTCCCTCTTTTTATGAAGCTTAGTTTTACTGGATACAAAATTCTTGGCTGACAAT TATATTGTTTATGGAGGCTAAAGATAGGACCCCAATTCCTTCCAGCTGGTAAGGTTTCTG CTGAGAAGTTAACTGTTAATCTGATAGGTTTTCCTTTATAGGTTATCTAATTCTTTTGTC TCACAGCTCTTAAGATTCTTTCCTTCATCTTGACTTTAGATAACTTGATGACTATGTGCC TAGGTGATAATCTTTTTGTGATGAACTTCCCAGAAGTTCTTCGAGCTTCTTATTTGGTTG TCTAGATTTCTAACAAGGCCAGGAAAGTTTTCCTCAATTATTTCTTCAGATAAGTTTTAT GAACTTTAGATTTCTTCATCAGGAAGACCAATTATTCTTAGGTTTAGCCATTTAACATAA TCCCAAATTTCTTGGAGGCTTCATTCATTTTTTAAACTTTTTTCTTTGTCTTTGTCTGAT TGGCTTTTAATTCAAAAGCTTTGTCTTTGAGCTCTAAAGTTCTTCTACTTGTCCTAGTCT ATTGTTGAAACTTACCATGGCACTTTGCATTTCCGTAGGTGTTTCTTTCATTTCCAGAAG TTGTGAGTATTTTTTCTTTATGATATCTATTTCTCTGGAAAATTTTCTTATCCATATCTT GTATTGTTGTTTAAATTTCTTTAAGTGGGTTTTCACCTTTCTTTGGTATCTCCTTGATTA GCTTAATAATAAATCTTCTGAATTCTTTATCTGGAAATTCAGAGATTTCTTCTTGGTTTG GATCCATTGCTGAGAAGCTAGTATGATCTTTTGGAGGTGTTATAGAACCTATTTTGTCAT ATTACCAGAATTACTTTTCTGGTTCCTTTTCACTTGGGTAGACTATTACTTAAAATTGTT CTTGGATTTATTTTTAATTGAACTGTGTTTTTTAAATTTAAAATATTTTCCCTCTTTAAC ATCAGGACAGTGTGTATTTTAGCCTAATTTGATTATTGGTGCTTGTAGGAGTAAAGACTC TGTATGAGATCCTTAGTTATAAAGAGTCATTGTGCACTGGCTTTCCCTGATGCTGGTTGT AGTAGTTACTGTCTTAGTGTGCTGGCAAGTTCACTGTCTCCTATGGAGTTGGAATGGCAG GGATCTCTTGCAGCTTATCTGGTTCTCTCATGGTGTACATTTTTATTTATTTATTTATTT ATTTTTTCCCAGTGTTTTATTTACTGACTTGATGATTCAGGCTTCAGGCCAATAGAGAAG GTATCCCTGAGTAGGCATTGGCTATGGCTAAGGCAGGTGGGTAAATGTAATACCCAACAG TGGGCTGAGGTCCCAGCCTTGATGAAGGTGGCTGGGAGACCTCTCAGTTAGATGCGCTGA GGTTTTATCAGGGTGAAGAGTAGAAGCTACCTCAGCTCCTCTGCCAGGTCAGCCAGAAAG CTATTCACCTCACAGCCTCACTCCTATCCCAGTGTTTTGGCTGTTCAGATCAGACAGCCA CCTCTTTTCATCTGTAGGAATGTTGATGTTCCAAGTAGGGAGGAACTGTGACTCTGCCTC TCACACAGCCTGAATCTAGGGTGTGCTCCTTCTCTGGGGCCGCACTCACCCTGGATTGTT CCAGACAGGCTGTCTATAGATGCCTCCATACTGTGTTCCTGTGGGGGAAGCCCCAGCTGT GTCTGCAGTGAAGTGCCAGAGGAAAACAAGGACCTCTTTTCTAAGGCCCTTCACAATCAC AGAGGATGCCTGCCCATTGGGCTAGAGGTGCAGACTTTCCCTACTGTGCCAGCACTGCAA TTGTGTTTCTGCTGTGAGAAACTACCCACCAGCAGAAAGATCTGGAACTCAAGACCTGCT CTTCAGATTCTTTTGTCCCACAGGGTGATCCCCTGATGTGGTGCACTCCCCCTTCCCTAG GGATGGGGCTTCCTGAAAGCCAGACTGCAGTATTGTTATTGCTCTTCTGGTTCTAGCCAC CTGGCAGGGCTACTAGGCTCTGGGGTGGTGCTGGGGAATGTTTGCAAAGAGTCCTGTGAT GTGATCTGTTTTCAGGTTTCCCAGCTGTGGATACCAGCACCTGCTCTGGTGGAAGTGGCA GGAGAGTGAAGTAGACTCTGTGAGAGTCCTTGGCTGTAGATACATTTAGTATGCTGGCTT TCTCAAATGCTGGTTATGCTAGCAGTGAAGTTGTCACGTGGACAGACTCAGGACCTCTGG TTAGTCAGGATGTTGCAGGCAGTAGAATTAGCTGTCGTTTTCTCCTTCCTGGGATCAGGG CTATTCTGTCATGAATTGCAGTCATGTCCTGAGTTGGTTGACCTCCAGCCAGGAGGTGGT GCTTTCAAGAGAGCACCACTGTGCTATTAGCAGTGGGATATACGCTTGCCCTAAGTTGGC CAGGGGAGGTATTCTGACTTTCAGGAGACAGGCAGAGCCATAAAACTCCCAAGGGTTTAT GTCTTTTGTGTTCAGCTACTAGGACAGGTAGAGAAAAACCATCAGGTGAGGGCAGGGTTA GGCAGGTCCAAGTGCAGACTCTCCTTGAGTGGGGCTTGCCATAACCACTGTGGGGGATGA GGAAGTGGTCCTCAGGCCAATGAGGTTATGTTCCAGAGGGGATTATGGCTACCTCTGCTG TGCAGTATAGATCACCAGAGAAGTGGGGGATAGCCAGTAGTGAAAGGCCTCGCCCAGCTC CCACACAGTTGGTGAGGCTGGTCTTGCTCCTGCAGTACCGTGCTAACTGCACCAAGTTTA GATCCAGGCAGCCTACTCAGGGATCTCAGACCTGCCCCAGGCCATAAGCTTCCCTGCTGA CAAAGCAAGCATGGCTTTCAGGCCACATCCCTCCCTGTCTGCCCACAATGTCAGCAGCTC CTGCACTCGTATCTGCAGCAACTCCCGTTAGCCCCCTGGATTCTGCTCAAGGAAGTTTGT GCCCAGTCGAAATTATCACAGAATCTAGTTGGAAGTTTCTTTCACCCTATGGCCCCTCCT TTTATTTGGCTGGCTGCCTTCTCAGAAGACCCCTGTGAGATACAGTCAGGAATGGCATTC CAGGGCCCAAGCTGGAGACTGGGAGTACCTTCAAGGTTCCTGTTGCTTCTTCTACTTTTG CACTTCATGCAGCTCCCTAAAGTCATTTCAGCTCTAGGTAAGGTTACAACCTTCTCTCGT GATCTGCATTTTCAGATTCCCCAGTGGGGATGTGTGTTTGGAGGCAGGTTTTCCCCCCTC TCACTCACACTTTGGGAACTCACGGCTTTTCACCTGTCTCGTGCAGTCTGCAGTGGCATG TCACTTCTTTCAAAGGATCTGTGAATTCTTCTGGTTTTCCTGTTACGTTCCTGCGGTGGT TCTTGAAGCAAAAGTCCACAGTGTGAGTCTCCACACACTGTTCTGTCCATCCAAGTGCGA GATGCACGTTAGCCCTGCCTCCTATCTCCCATCTTCCTGTTTTTGCTTTTTTCAATTTTG ATGAGTTTAAAATGTACTTGTAATACTTTTTACCAGTTTTTATTAGTGTGAGGTTAAGGT CATGCATACTTACTTCCACTTTACAAGTATTTATGATAAACTTTGTATATCAGGGATTGA CAGACTTTTCCTTAAAGGCCCAGATAGTAAATATTTCAGTCTTTGCTGGCTATATGTCTC TGTCATAGTACTCAACTCTGCCATTGTGGTACAGAAGCAGCCATAGACAATATAGAACAA ATAAACATGCCTGTATTCCAACACAACTTTATTTAGGGACATGGAAATTTGGATTTTATA TAATTATCATATGTCGCAAAACTTTATTCTTCTTTTGATTTTTTTTTTCACCAAACATTT AAAAATGTAAAACCCATTCTGAGCTCATGGGCTGTACAATAACAGATAGGTCACTGGATT TGTCCTATAGGCCATGGTTTGCTGATCCCTGTGCTCTATGAATCCTGCTTTATCAGGTAC TATGGGGCAAGATAGAAGTAGCATCCAGGGCAGCTCTGTGCTTGCAGTCTAATTGAGGAG CTGAGATGTTGTATACATACTTTAGAGCATTGGGTTATAAAGTAGTACAATAGGCACAAC AGAGTTGGTAGCTCTGACAAGTGAGTTTGAATGCTCCATAGCAGAGCAGGTCAGTGGAGG CTGAAAAGTCAGGGAAAGCAGGATGAAAGAAGGGCTTGCAATCGGAAGCATGCAGATAAT TTGGAGGTTTCCATTTGAACATGAGATCGCAAATGATGAATGCAGTCTACTTACTGCTAT TCTCGGCACCCAGAATAGGGCCTGGCATAAAATGTGTTCTCAGTAATTATTTATTGAACA AAAAAATATGTTTTAAGGCACAAACAGAGTCCATCACACTAAAATGAAGTATTTTTGATG CACTAAAAAAGTCAATGAATGTCAGGGCCAAGGATTTAGAATTTATCTTTTATATAGTGA GAGATTTTTGACCACAGGGTTAATGATAAAATTATATTTGGGGAGGTTTAATCTGCCAGC GGTTTATGGGACTTCAGTTTACCTAAATTGAATCTAGCTTTTAAATACCAATCTCACTTT CTAGTACTAATTTCACTTTAAAATTCTTACTTGAACTTGAGCCAACAACAAGAAAAGAGA GAATTTTTGTTTACATATGCATGCTATACAACCACTGACTCATTATCAGTTGGTCTTGGG TTTGGCATTAACTAAAAGAGTCACAAGACACAATATGTTCTGACATATTTATCAGTTTAT TTCAAGCAACACAGCACACAGCCCAACAAAAGTTGCAACACATAGATGCACTGCACAGAG CATGCTAGGCTGCAAGAGATGAACTGAAAGTGGGTGCAGACAACATTAACATAGGAAAGC TGTCACCTCCATTCCTTAGCCATCTTTTATAGCATGCTTATCACATGGCTTATCTGGAAG TAGGTGCAAAATCACAGCTCTCCCTGTGTAGCTACAACTCACCAATCAAGCCTTCAATGA ATGATTCTGGTAATCAAATACAATCTCACCACCTAACACTGCACGTTTATCTTGACTTTT ATATTCCAGATTAGTGTCGCATGAGATAAATCCAACATCATATCACCAGAAGTCCCTGAG TCTGGGCTTAGACTTTAAATCAACAGTTAGCCATTGACCCACAACTTTATCTCCCCAGAG ATTTTTTTTCCATCTCTAAAATCCAAATATTAGCTTTTGACCCTCGAAACAAACGAAGAG CAATTGCATAGGTGGAAAACATAGCTGTTTTAGCCTGAGCATTGATGCATTTCATTTCCA GTAATAGAACAAGGGGTTTGGAATCACTTCGATTTCTCCTGAAGCATCTTCACAGGGTGG CTTCATTTCTTGGTTTAAAATTCATACTGAGGTATAGGAGTCACACTGCGTTCTGAAGTG ACATAGAATGCTTTTTTTGATCATCCAACACTTTAGGTAATCCAACACTTTAGGTAATTT ATGCTCTTAATCTCCTCTATTTGTCTAGATGTTAGAGTTAACTATAAAACCAATACTCAC CCTCCCTCATATTTCTTTTTGCCCTTCATTCCGCTTCCCTGGAGTGCACCTTTTCTCCAG CTCCTTTCTGACTTCCCAAATTCATGCTTAAATAGGAAAGGCACTTGACTTTCTAGTGTC TTCTCCTGGCTCTTTGATAAAAGCCTTGGGGCTCCCAGCACATGGTTCGAAAGTCACAAC TGAAATGTAGTGTCCTCCAGTGTTGTCCCTACCCTCCTTCCTCTCATTGCCCTTGCTCTC CCCAAGTGATATATAATTCACTCCCGTGGTTTTAATTCATATCAGTATTAATGTCATCTG TATTCTAGCTTCTGTACCTGCACTCCAAACTCATACTTCTACCTGCCCATTGCCCTTGGA CCCCCCACTGGCACCTCAACTCAACCTGCTCAGGTCAGATCTTATCATCTCTCTTTAATA TTCCTCATCCTCTGTTCTCTGGCTGGTTAGATGGTTTTATCAACCGGAAACTATATTCTC AGTCTTAACTTTCTCAAAGCCCACTACATTTGAATTCTCTGACGGTTCTTGCTTCCACCT CACATTAGCTCTTAAGCCTATATGCTTTTATCTTTATCTGCTGTCACCACCACAAGCCAA CATCATCTTTGCCTGGATTATATAACAGATCCCTTGGGTGTCTAAAGATGTATTCTTAGG AGTATGTGAGTTCTCTAAAGATAATAGTGTATGCCACTTTGATTAGGTGATAATATAATA TAAAACAATATGTACACAGATTGTAAAGCAACCAACGTATAGTCAAGAATGTTATAAAAT TTCAATGTCTGAATTTGTATTTAGGAATAGTTTTCCACAAAGTGAAGAACAAAGTAGTGC TTCACAGCAGTTGTATTAAAGAAAATAATGGAATAATTGAGTAATTTCACTGCATATGGT AGACTTGGCATGCAAACTTGAGTTAGGACCTGTACCTGAACAATCAGCATATACTCACTT TTTGGGCAGTATGGCCATCTATATATAAACCACATGTAAATTCACCCTCAGTTATAGTAT AAATTAATCATTTTTTTCTATTTTCAGTTTTAGTAATTATTGGATTATAATCTCATCTAT TAGCTTTATCTTCAAGTGAACAAGAATTTTAAAATTGGCATTGAAGAATTATTTGAATAC AGAATTTTGGTTAAGTTCTGATTATTGTGAATTGGCAAATGGAGCATTGAAGATACTAAT TCCATTTTGTCCAACATACTTATGTGAGCAGGTATTCTCTGCACTAATATTGTTGAAGTA AGAATACAGAAACAGATAAAATATAGATACCAAGATTCATCTTTTTATTATTAAATCCAA CATGCACTGTTTTATTTCAGCAAAGCACATCATCAATCACATCAAAGTTATGTTGTTACT AAGACTTTTTAATATTTTTTGTTTTAAAATGGAATTAAAGCTATAAGCCCAAGCACTTAA AACCAGTTACTAATTACATTTTAATGAAAATATATAAGGCAAGCCTGGGGATTTATAAGA ATTTCTTGCCCTTTGAAAAGGGTCTGTGTATTTTTCAAGGAACTGCTTTAATTGTAAGGA AAAACAAATTCTGTTGTATTTATGTTCTTTTTATTGTATGTAATTTTATTCTTGCATTCA AAATGATTTATAATTTCTTTTTTTTCTCAGAACAAATGTCAAGGTCTCGTGTTCGACATA GTCACAAGCCAGATCTTTGACATCATCATCATAAGTCTCATTATCCTAAACATGATTAGC ATGATGGCTGAATCATACAACCAACCCAAAGCCATGAAATCCATCCTTGACCATCTCAAC TGGGTCTTTGTGGTCATCTTTACGTTAGAATGTCTCATCAAAATCTTTGCTTTGAGGCAA TACTACTTCACCAATGGCTGGAATTTATTTGACTGTGTGGTCGTGCTTCTTTCCATTGTT AGTAAGTAAAATCAGCAGTCAGAGGGACTTTAAGAACCAGAAGTAAGTTTGTAAATCTTA TCATTTTTTGAAGTTTGTTCAAACTATCCACAAAGCAGAAAACTGGGCCAAGTGTACTTT CTGAAAGAATAGACAGGGGTACTAATGCCATTCTCTACTGGGAAGTTGCTAGGAGATAGA GAGTAATTTCTGTTCCCTTAACTCACTACACAACTGAAATAGAGTTCAATAATCATGCAG CTAATGTATTCAATGGAAATAGACAAAATTAAAATGACTCAGAAGTTTTTGTGGTGGTAA CCTGATTTATTCAGATAATTGAGTCAAAGTTCTACCTTTCCTGTGCAACTATAAGCCCTA TGAGCATTTTTTAGTTTTAAGGATTTACTGCCCATGTTTTTTCCTTTGTTCAAAATCCAC ATGGGTGCCAGATCACAATTATTGTGCCCCTGTGGCACTGCTGTGCCCCCTTTCAACCCC TATACAAGGCCCCATGGCCCACTCTAGATTGGGCTTTCAAGAGTATATCATTCAGACAAT CAAAGCTTTTTTTCTATTCTATATCCCATGTTAACTTAAACCACAGTGTGAACTGTGCTT GTCTCAGATTCTTTGACCCCGCTTACCAACCAGCCTCATTCTCTAATCAGTATATTTTTC CTAATCACACGCAGGGTTAACTAAAAGGCTTATAAAAGAATACAAAAGAAAGAAGGTTAT TACCATACTCCTTCTTATAAAACACACACATACACACATCAACTTTAAAATTAAATTCTT CATGGCTAAACCATAAGTGCTTACTCTGGGTTTTTGAAGTCTGGTTTTAATTAAACTGTT AACTTGACATTTTATAATTATGTAAAACTGTCCAGGAGCCCTTCTCATAGTTCAAAACAC AATGAAGACAAAGAAAACTGATTTTCCAAATGGAAGCTTTTTTGCGATCTCAGAAAGAAT GCATCAAACAGATATATTGGATCAAGATATTTTGGTTCCTAATGCTACTGCAGCATTGCA GATGTTTCAGAGAACAACAACAAATATGCTACAGTTCATAGTGTGTTCTGGTTTCTAAGC CATGATGTTAATGTAGATAAAGCTTATCAATAGATTCTTCCATGAGTTTACAGTTAAGTA GACCTTTAATTTTTATTAGTCAATTCCAATCACAAGTCTTGGTGAAATTGACAGCAGCAA AAAAAAAAAAAAAAAAAAAAAAAAGGGCTAGAAAATGGTGGTCAGATGTCTGGGGAGGCC CTCTAAGAACTCAAAGAGCCCTTCCTCTGATGAGAAAAGAGAAGCTCATTCATTCCAACA GCTCCTTCCATGCAGTGCCAGGCATCATGCACTTTGCGGAAATGTTTCTGACTTTTGTCC TTTGAACAACTGCTTGGCAATTGCAGTTATACATGCTGTATGGCATTTGCCCCAGTGGAT GAGAAAATCTACATATTAAGCCAAATAATACTCAGCGGTAGGATTAATCCATATATTAAT CCTACTTACTAATGTGGACAGAGTTTTATATGCCTTTGCCAATATTTCTGCCGTCCAACG CTTTAGCCTCAGTACTTTTCCAAGCTCTTTCCTAAGTTTTTGGAGAAATGCAAAATGAGT GTGAGTTGAAATGAATCTCTCTCTAGGAGTTAACAATCTAGTTGAGGTATTACGGCTTAC TCGTCTTAAAGTGCCAAAAAAAGATAGGGAAATTAATTGAACACCAAATCACATGGTGCA AAGCAGAAATGAGCTCAGAGAAAGAACAGATTACTATGGGCTTGATCAGCCCAAGAAATA TTCATAGAGGAGGTAGATCTTAAATGATGGGCCTCCTCCAGTTGTTGCTGAATTGGCACT TAAGAAACTTTACATCTTTCCAGGGTTGACTTTCCAATTAAAGTATGAATTAAACTTCTT TGGCACAATTGGGTAGTCAGTGAAGTATATAATCAACTAGTTCTCTTTAGTCATTAGGTG ACAGCAGAGCATTCTGCTGCCCATGAACCCTAATAGACCCCTAGTGACCTTACAGGACAC TCAAGGTCATTAGGGGTCATGGGCACTCAAATACTGCCCATGACCCCTAACAACCCTAGA AGACTCTCAAAGCAAGAACAAGGCCATGTCTACACAAGACAAGCATGTTCCAACCATTCT ATAATGTTTAGGCCAAAATGTGAATTGTCTTAAATCCTTGTATCACTTGCCTGCAGAGCA GGCAGGGCGACATTTTAGGTCAAATATTCAGCTGTTTGATCAAACCTCTGAAATGACATT TATGGGGTTTGCAGCAACAGAGAAAGTATCTCCAAATTGTGTCCCTTCAGCTTTTTGAGT TTGGATCTCAGAAGTCCTTGGAATGAGGATGACAATTATGAAAGATCCATGGGCAGCTTC AGCCTAAAGGTGAGCATTCCAATGCTACCATTTCCCTCTAGCCCTACAGACATTGTGGCT CTTTCAGCAGATAAGCTGTCCTCACTTGTAGTAGGGCTGTATTTAAATTCTTCTACACAC AAATGTAGAAGAACTGTATGGCCCTTGGAAACTATTTTCCAAATTAAGACACAGATAGGT ATTTTCCCTGATAGTGGTAAAAGAAATTCTTAGTTTTGTACATAAGCTACAAAGTATTGA TAAAAAGTTGGCCCCCATAGGATTCTGAGGCAGGAGACAGGCTGGATCTGAAACCAGTGT GCCTTCTGGAAACTTATTTACTTTTCTCCTTCTCCTGAGGTACAATGATTTCTACCTTGG AAAATCAGGAGCACATTCCTTTCCCTCCGACGCTCTTCAGAATTGTCCGCTTGGCTCGGA TTGGCCGAATCCTGAGGCTTGTCCGGGCTGCACGAGGAATCAGGACTCTCCTCTTTGCTC TGATGATGTCGCTTCCTTCTCTGTTCAACATTGGTCTTCTACTCTTTCTGATTATGTTTA TCTATGCCATTCTGGGTATGAACTGGTTTTCCAAAGTGAATCCAGAGTCTGGAATCGATG ACATATTCAACTTCAAGACTTTTGCCAGCAGCATGCTCTGTCTCTTCCAGATAAGCACAT CAGCAGGTTGGGATTCCCTGCTCAGCCCCATGCTGCGATCAAAAGAATCATGTAACTCTT CCTCAGAAAACTGCCACCTCCCTGGCATAGCCACATCCTACTTTGTCAGTTACATTATCA TCTCCTTTCTCATTGTTGTCAACATGTACATTGCTGTGATTTTAGAGAACTTCAATACAG CCACTGAAGAAAGTGAGGACCCTTTGGGTGAAGATGACTTTGACATATTTTATGAAGTGT GGGAAAAGTTTGACCCAGAAGCAACACAATTTATCAAATATTCTGCCCTTTCTGACTTTG CTGATGCCTTGCCTGAGCCTTTGCGTGTCGCAAAGCCAAATAAATATCAATTTCTAGTAA TGGACTTGCCCATGGTGAGTGAAGATCGCCTCCACTGCATGGATATTCTTTTCGCCTTCA CCGCTAGGGTACTCGGTGGCTCTGATGGCCTAGATAGTATGAAAGCAATGATGGAAGAGA AGTTCATGGAAGCCAATCCTCTCAAGAAGTTGTATGAACCCATAGTCACCACCACCAAGA GAAAGGAAGAGGAAAGAGGTGCTGCTATTATTCAAAAGGCCTTTCGAAAGTACATGATGA AGGTGACCAAGGGTGACCAAGGTGACCAAAATGACTTGGAAAACGGGCCTCATTCACCAC TCCAGACTCTTTGCAATGGAGACTTGTCTAGCTTTGGGGTGGCCAAGGGCAAGGTCCACT GTGACTGAGCCCTCACCTCCACGCCTACCTCATAGCTTCACAGCCTTGCCTTCAGCCTCT GAGCTCCAGGGGTCAGCAGCTTAGTGTATCAACAGGGAGTGGATTCACCAAATTAGCCAT TCCATTTTCTTTTCTGGCTAAAATAAATGATATTTCAATTTCATTTTAAATAATACTTAC AGAGATATAAGATAAGGCTACTTGACAACCAGTGGTACTATTATAATAAGGAAGAAGACA CCAGGAAGGACTGTAAAAGGACATACCAATTTTAGGATTGAAATAGTTCAGGCCGGGCGC AGTGGCTCATGCCTGTAATCCCAGCACTTTGAGAGGCCAAGGCAGGTGGATCACGAGGTC AAGAGATCGAGACCATCCTGGCCAACATGATGAAACTCCGTCTCTACTAAAAATACAAAA ATTAGCTGGGCATGGTGGCGTGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGA GAATCGCTTAAACCTGGGAGACGGAGGTTGCAGTGAGCCAAGATCGTGCCACTGCACTCC AGCCTGGTGACAGAGTGAGACTCTGTTTCAAAAAAGAAAAGAAAAGAAACATGGTTCAAA TTATATCTAAACAAAAAAGAATAAGAAACAAAAAACACATTAAAATTTTAAGTTGTATTT TCTATGTTTCTAGATACATCATTTTTGTTTGATATTTTCCTGATGCAAGTATGTGGTTTA TCACATGTAGCTCTTTTGCATGCTAAATGAAAATTCAAAACTTGCCAATAAATGAATAGC TTATTGCAGACATTTTTTACCAACATTAATTATTTTGGGTTTGTTTAAAACCTAGAGGCA CAATCTTGACTTGTCAATTACTACCCTTTCACAAGCTACCATCTCAGATATATATATATA TATAAATTCAATAAAGCTTTCTGTTTGTGTTCCTTTCAGGTAGTACACTAATATTCCCCT TTTGTTCAGTGGTTGTGTGAGTAGTGGTTTAAATGTGGTGCTTTGCTTGCAATTAAAGAC TAGAACCAGTGGTGTATCTGACAAGTGTTTAACAACTGACTGTTCAGGAAAAAGAAAGCC ATGCTTTATAGCATTCACTAATCTCTGTGGTGTGAGTACTCCCATTATGGCTGATTTCAA GCTATCAACATGTTACTGGACGTGGAGTTGAGAAGAGATGCTCATAATAAGTTCTTGAGA GCTGGCTCAAGCTGACTGCAGCACATCACTTGCTGAAACTGAAAAGAGAGCCCCACTTGG CTGGTTAAGATATTTGCAGTGGGCACCTTGGACAGAGAGAGTACCCTCTTAGCAACACGT GGGGCACGGGGTGAGAGATCCATAGACATACTGCTTAACCAGTGATCTTAGCCATTTGAT GAGCCCTATTTACAGTTTTGGCTGAATGTCTGTTTCAAAGAAACACCCAGACCTATCAAA AGGGCTTGGGAGCAGGAGGAGCCCAGGAGTTCCCTAAAAACTGGAGATGCCAAGAGGTCA AACAGGCATTAGGCTTTCAGTCCCCCAAAAGGCTGGGCGAGGGGCAAAGAACCTACATTG TGATCATTGAGGAGTGGAGGAAAACCTCATAGAGATAGCCCTGGAACCCTGTATCTCAGA CAGGTGAGAAGAACTGATGTGTCTGTCAGCAGCCTGGAGTCTGAGGTCGGGCCCTCTGAC CTGCCCCACACTGCCCAGCAAAGGGAATGGCAGCACTGGATCCTGTGGTGGGCTGAGAGG TGATTATTTTCAGTGTGTAAGATCTTACCATGACTTGATGTATTCATTTCAAGGGCCTGA GTTTTGTAGGCAGCCTTGTGCTCTGAAGCATTGGTAAAAATAGTGATAATATCATTATCT AATATTTATTTAGTGGTTTCTGTGCATCAGGCACTGGCTAAGCATTTGTCATGAATGATC TCTTTTGTACCTCGTACCAACCCAATGAGGAAGATATACCCCATTTAAAAAGGAGAAAAC TGAGGCTCAGAGAAGTTGAGTGACCTGCATACAGTCATGCACCTGGTGGAGCCCGAGCCT GTGTGCCATGATGGGAAGGGGAAGTGGGTGGGCTCAGGACCAGGGAAGCTTTGAGATGGG CTACCAGGGCTCCTGTGCAGGACTGGGAGGGCC

Example 5: VEGF-A, PDGF-B PATHWAY & ANG-2

VEGF-A is also referred to as VEGF, Vascular Endothelial Growth Factor A, MVCD1, VEGF-A, Vascular Endothelial Growth Factor, Vascular Permeability Factor and VPF. Human VEGF-A binds to the human FLT1/VEGFR1 and KDR/VEGFR2 receptors. Human isoforms, including VEGF-165 and VEGF-145, have been observed.

Vascular endothelial growth factor (VEGF) is a highly specific mitogen for vascular endothelial cells. Five VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene. These isoforms differ in their molecular mass and in biological properties such as their ability to bind to cell-surface heparan-sulfate proteoglycans. The expression of VEGF is potentiated in response to hypoxia, by activated oncogenes, and by a variety of cytokines. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis. In vivo VEGF induces angiogenesis as well as permeabilization of blood vessels, and plays a central role in the regulation of vasculogenesis. Deregulated VEGF expression contributes to the development of solid tumours by promoting tumour angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis. Consequently, inhibition of VEGF signalling abrogates the development of a wide variety of tumours. The various VEGF forms bind to two tyrosine-kinase receptors, VEGFR-1 (fit-1) and VEGFR-2 (KDR/flk-1) in humans, which are expressed almost exclusively in endothelial cells. Endothelial cells express in addition the neuropilin-1 and neuropilin-2 coreceptors, which bind selectively to the 165 amino acid form of VEGF (VEGF165).

VEGF is a key regulator of angiogenesis, and its expression in producing cells is regulated by a plethora of external factors. Cytokines, growth factors, and gonadotropins that do not stimulate angiogenesis directly can modulate angiogenesis by modulating VEGF expression in specific cell types, and thus exert an indirect angiogenic or anti-angiogenic effect. Factors that can potentiate VEGF production include fibroblast growth factor 4 (FGF-4), PDGF, tumour necrosis factor a, transforming growth factor β (TGF-β), keratinocyte growth factor (KGF), IGF-I, interleukin 1β (IL-1β), and IL-6.

Angiogenesis has a causal role in many diseases, including neovascular age-related macular degeneration (AMD). Identification of key regulators of angiogenesis, including vascular endothelial growth factor (VEGF), fibroblast growth factor 2, pigment epithelium-derived growth factor, angiopoietins and extracellular matrix molecules, has facilitated the development of novel therapeutic agents that target the underlying pathological angiogenic process. Among these, VEGF serves as a “master switch” for many ocular neovascular conditions through its promotion of endothelial cell proliferation and survival, vascular permeability and ocular inflammation. Anti-VEGF agents are now available, such as aflibercept (EYLEA™), bevacizumab (AVASTIN™, ranibizumab (LUCENTIS™) and pegaptanib sodium (MACUGEN™), an aptamer for neovascular AMD. Unlike bevacizumab, which binds all VEGF isoforms, pegaptanib targets only VEGF165, the isoform responsible for pathological ocular neovascularization.

Flt-1 (VEGFR-1; Unitprot ID: P17948) is one of the two human receptors for vascular endothelial growth factor. The other being KDR (VEGFR-2; Uniprot ID: P35968). The fit-1 protein consists of an external domain containing seven immunoglobulin like domains, a transmembrane region and a cytoplasmic region containing a tyrosine kinase domain. In contrast to other members of the receptor tyrosine kinase family, the kinase domain of fit-1 is in two segments with an intervening sequence of ˜70 amino acids. The biology of the VEGF receptors has been reviewed (Neufeld et al., (1999) FASEB Journal. 13:11-22; Zachary (1998) Experimental Nephrology. 6:480-487) and the tyrosine phosphorylation sites have been identified (Ito et al., (1998) J. Biol. Chem. 273:23410-23418).

It is thought that flt-1 may be important in regulating the tissue architecture in developing vasculature while the second VEGF receptor (KDR, VEGFR-2) mediates the mitogenic and angiogenic effects of VEGF in endothelial cells. Evidence to support this theory has come from knockout studies in mice (Fong et al., (1995) Nature. 376:66-70). VEGF and its receptors are over expressed in many tumour types and blocking of VEGF function inhibits angiogenesis and suppresses growth of tumours while over expression of VEGF enhances angiogenesis and tumour growth (Skobe et al., (1997) Nature Medicine 3:1222-1227).

The fit-1 cDNA (EMBL Accession Number X51602, 7680 bp) encodes a mature protein of 1338 amino acids. The structure of the murine fit-1 gene has been determined (Kondo et al., (1998) Gene 208:297-305) and has been used to predict the intron/exon boundaries within the human gene. The promoter region of the human gene has been characterised (Ikeda et al., (1996) Growth Factors. 13:151-162; Morishita et al., (1995) J Biol Chem 270:27948-27953; EMBL Accession Number D64016,1745 bp). The fit-1 gene, which is organised into thirty exons, has been localised to chromosome 13q12 (Rosnet et al. (1993) Oncogene 8:73-179). See EP1130123A2.

Diabetic Retinopathy (DR)

Diabetic retinopathy (DR), a micro-vascular complication of diabetes, is a main cause of blindness in adults. It is well established that DR is determined by both genetic and environmental factors. The longer duration of diabetes, poorer control of blood glucose and elevated blood pressure are the major risk factors in the development of DR. However, genetic factors also play important roles in the pathogenesis of DR. Over the past three decades, the number of people with diabetes mellitus has more than doubled globally, making it one of the most important public health challenges to all nations. DR occurs in 75% of patients with type 2 diabetes mellitus and almost all patients with type 1 diabetes mellitus within 15 years of the manifestation of diabetes. Evidence supports an important role for genetics in determining risk for DR.

VEGF-A has been implicated as a major contributor to the development of DR. VEGF-A could induce the earliest changes in DR including leukostasis, blood-retinal barrier breakdown, and macular edema and neovascularization in progression of DR. The VEGF-A gene is located on human chromosome 6p21.3 and consists of 8 exons. The genetic variants in the VEGF-A gene are suggested to influence levels of VEGF-A protein expression. To date, many studies have investigated the associations between polymorphisms in the VEGF-A gene in humans and DR. −634G/C (+405G/C or rs2010963) polymorphism in the 5-untranslated region and −2578C/A (−2549I/D (in high linkage disequilibrium with −2578C/A) or rs699947) polymorphism in the promoter region of the VEGF-A gene have been investigated.

Age-Related Macular Degeneration (AMD)

Age-related macular degeneration (AMD) is a neurodegenerative disease that leads to visual impairment and accounts for half of all cases of registered blindness in Western individuals older than 65 years of age. There are approximately eight million people in the United States with symptoms of early or intermediate AMD, of whom approximately one million will develop advanced AMD within the next five years. AMD is estimated to affect about 50 million people worldwide, and an increase in aging populations makes AMD a significant public health concern and a major focus of research efforts. AMD is a clinically heterogeneous and genetically complex disease, with multiple environmental and genetic risk factors involved. While epidemiological studies have linked cigarette smoking, alcohol consumption, light exposure, diet, drugs, and high blood pressure to the risk of AMD, familial aggregation and twin studies have suggested that genetic variation may also play an important role in the disease.

Anti-VEGF therapies are now regarded as the standard of care in the treatment of neovascular AMD. Two of the most widely used treatments are ranibizumab and bevacizumab. Ranibizumab (Lucentis®, Genentech, CA, USA), a US FDA-approved anti-VEGF therapy for AMD, is a recombinant, fragmented, monoclonal antibody that binds to VEGF. On the other hand, bevacizumab (Avastin®, Genentech), although not FDA approved for ocular treatment, is a full-length monoclonal antibody reportedly with similar function as that of ranibizumab. Bevacizumab, FDA approved for metastatic colon cancer, has been given as an off-label treatment for ocular angiogenic disorders. An individual's genetic variation may affect the treatment response in both of these drugs.

Polypoidal Choroidal Vasculopathy (PCV)

A subset of wet AMD, termed polypoidal chorio-vasculopathy (PCV), has been identified as a clinical entity. PCV is important, as it may be the main cause of vision loss from AMD in certain Asian and

black populations. The distinctive clinical features of PCV include one or more haemorrhagic pigment epithelial detachments (PEDs), extensive exudation, and disease outside of the macula including the periphery. There is a predilection for peripapillary ‘polyps’. Patients are also characteristically younger.

There is increasing evidence that Asian patients with neovascular AMD have a variant of AMD, ie, polypoidal choroidal vasculopathy (PCV), which is characterized by polypoidal lesion with inner choroidal vessel abnormality. PCV is more prevalent in Asian subjects, reportedly accounting for about 50% of neovascular AMD compared to 10% in Europeans. Studies have shown that anti-VEGF therapy (ranibizumab) in combination with photodynamic therapy (PDT) was associated with a more favorable outcome compared to ranibizumab monotherapy.

Retinal Angiomatous Proliferation (RAP)

Another variant of AMD in the spectrum of occult CNV is retinal angiomatous proliferation (RAP), which represents about 12%-15% of neovascular AMD. It is associated with proliferation of intraretinal capillaries with retinal anastomosis or CNV.

Diabetic macular edema (DME)

Diabetic macular edema (DME), the most common cause of visual loss in subjects with diabetes, is a separate classification assessed independently from the DR spectrum, because it can develop at any stage of DR. The pathogenesis of DR and DME is thought to be related to the loss of pericytes, thickening of basement membrane and endothelial cell loss, leading to microaneurysms, blood-retinal barrier breakdown, increase in inflammation and vascular leakage. There are several treatment modalities for DME. The goal of laser treatment of DME is to reduce disease progression by targeting areas of leakage on the retina. Intravitreal triamcinolone acetonide (IVTA) has also been shown to significantly reduce DME, with maximal action at one week and lasting 3-6 months.

Retinal-Vein Occlusion (RVO)

Retinal-vein occlusion (RVO) is the most common retinal vascular disorder after diabetic retinopathy in the elderly and is often associated with systemic disorders, such as hypertension, hyperlipidemia, diabetes mellitus and/or arteriosclerotic vascular disorders. It is classified as branch retinal vein occlusion (BRVO) or central retinal vein occlusion (CRVO), depending on the site of occlusion and further divided into ischemic (non-perfusion) or non-ischemic (perfusion) RVO, each with differing prognosis and treatment. Macular edema and retinal neovascularization are the two most common causes of visual impairments; thus, ocular managements with laser photocoagulation, intravitreal injections of glucocorticoids or anti-VEGF agents, as well as other surgical or systemic therapies, have focused on these two sequelae.

Retinopathy of Prematurity (ROP)

ROP remains one of the leading causes of childhood blindness. In late stages of ROP, neovascularization of abnormal or pathological vessels arise, due to retinal immaturity, and lead to retinal traction, detachment, hemorrhage and funnel configuration, eventually resulting in poor vision. Neovascularization is mainly driven by VEGF, and currently, the recommended treatment for Type-1 ROP is peripheral ablation by laser.

Angiogenesis & Angiogenesis Inhibitors

Angiogenesis is the formation of new blood vessels. This process involves the migration, growth, and differentiation of endothelial cells, which line the inside wall of blood vessels. The process of angiogenesis is controlled by chemical signals in the body. These signals can stimulate both the repair of damaged blood vessels and the formation of new blood vessels. Other chemical signals, called angiogenesis inhibitors, interfere with blood vessel formation. Normally, the stimulating and inhibiting effects of these chemical signals are balanced so that blood vessels form only when and where they are needed. Neovascularization is the formation of functional microvascular networks with red blood cell perfusion. Neovascularization differs from angiogenesis in that angiogenesis is mainly characterized by the protrusion and outgrowth of capillary buds and sprouts from pre-existing blood vessels.

Angiogenesis plays a critical role in the growth and spread of cancer. A blood supply is necessary for tumours to grow beyond a few millimetres in size. Tumours can cause this blood supply to form by giving off chemical signals that stimulate angiogenesis. Tumours can also stimulate nearby normal cells to produce angiogenesis signalling molecules. The resulting new blood vessels “feed” growing tumours with oxygen and nutrients, allowing the cancer cells to invade nearby tissue, to move throughout the body, and to form new colonies of cancer cells, called metastases. Because tumours cannot grow beyond a certain size or spread without a blood supply, research is focused on trying to find ways to block tumour angiogenesis, with the idea that angiogenesis inhibitors will prevent or slow the growth of cancer. Similarly, ocular diseases and conditions, such as AMD and DR are associated with angiogenesis, and inhibitors are useful for the treatment or prevention of these too.

Thus, anti-VEGF (ie, VEGF-A) ligands and antibodies of the invention are useful for treating, preventing or reducing the risk of one or more of such ocular conditions and cancer (eg, solid tumours).

Angiogenesis requires the binding of signalling molecules, such as vascular endothelial growth factor (VEGF), to receptors on the surface of normal endothelial cells. When VEGF and other endothelial growth factors bind to their receptors on endothelial cells, signals within these cells are initiated that promote the growth and survival of new blood vessels. Angiogenesis inhibitors interfere with various steps in this process. For example, bevacizumab (Avastin®) is an anti-VEGF monoclonal antibody that specifically recognizes and binds to VEGF. Aflibercept (Eylea®) is a VEGF receptor-Fc fusion that binds VEGF. Other angiogenesis inhibitors, including sorafenib and sunitinib, bind to receptors on the surface of endothelial cells or to other proteins in the downstream signalling pathways, blocking their activities.

In addition to, or alternative to, targeting human VEGF-A to address ocular conditions or cancer (or angiogenesis or neovascularisation generally), it may be useful to target the platelet derived growth factor pathway (eg, targeting PDGF-B and/or PDGFR-B), or the angiopoietin-2 pathway. Further details are provided below.

In an example, the invention provides a method of treating or reducing the risk of a VEGF-A-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human VEGF-A protein. The invention also provides a corresponding ligand.

In an example, the invention provides a method of treating or reducing the risk of a PDGF-B (ie, platelet derived growth factor-beta)-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human PDGF-B protein. The invention also provides a corresponding ligand.

The present invention provides anti-VEGF-A ligands; and VEGF-A-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of VEGF-A, in particular human VEGF-A or its ligands and in screening assays to identify other antagonists of VEGF-A activity. Some of the ligands of the invention are useful for inhibiting VEGF-A-mediated activities.

In an example, the invention provides a method of treating or reducing the risk of a PDGFR-B (ie, platelet derived growth factor-beta receptor)-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human PDGFR-B protein. The invention also provides a corresponding ligand.

The present invention provides anti-PDGF-B ligands; and PDGF-B-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of PDGF-B, in particular human PDGF-B or its ligands and in screening assays to identify other antagonists of PDGF-B activity. Some of the ligands of the invention are useful for inhibiting PDGF-B-mediated activities.

The present invention provides anti-VEGF-A ligands; and VEGF-A-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of VEGF-A, in particular human VEGF-A or its ligands and in screening assays to identify other antagonists of VEGF-A activity. Some of the ligands of the invention are useful for inhibiting VEGF-A-mediated activities.

The present invention provides anti-PDGFR-B ligands; and PDGFR-B-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of PDGFR-B, in particular human PDGFR-B or its ligands and in screening assays to identify other antagonists of PDGFR-B activity. Some of the ligands of the invention are useful for inhibiting PDGFR-B-mediated activities.

Anti-VEGF-A ligands (eg, antibodies and anti-sense RNA) have been developed based on targeting and neutralising so-called “wild-type” human VEGF-A, which is a commonly-occurring form. While such therapies are useful for human patients harbouring this form of human VEGF-A, the inventor considered it useful to investigate the possibility of targeting rarer—but still naturally-occurring—forms of VEGF-A amongst human populations. In this way, the inventor arrived at insight into the natural occurrences and distributions of rarer human VEGF-A forms that can serve as useful targets (at the protein or nucleic acid level) for human treatment, prophylaxis and diagnosis pertinent to diseases and conditions mediated or associated with VEGF-A activity. This particularly provides for tailored therapies, prophylaxis and diagnosis in humans that are devoid of the common VEGF-A gene or protein.

The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in activity and/or conformation of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to more effectively tailor medicines and diagnosis of patients. The invention, therefore, provides for tailored pharmaceuticals and testing that specifically addresses rarer VEGF-A polymorphic variant forms. Such forms or “alleles” (at the nucleotide level), comprise one or more changes at the nucleotide and amino acid levels from the corresponding common form nucleotide and amino acids sequences, ie, there are one or more non-synonymous (aka “missense”) changes at the nucleotide level that translate into one or more corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting such rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention. In particular, human VEGF-A variations are correlated with increased incidence or risk of VEGF-A mediated diseases or conditions, such as ocular conditions or cancer. Additionally or alternatively, VEGF-A variations are correlated with improved responses following treatments for ocular conditions. The inventor analysed such variations and devised the collections of variants in Tables 18-20. As shown in those tables, the inventor also assessed variation in other human targets that also correlate to incidence or risk of diseases or conditions, such as ocular conditions or cancer, or improved responses following treatments for ocular conditions. In this respect, therefore, the invention provides various aspects and concepts as set out below in this example.

The inventor saw that there is significant industrial and medical application for the invention in terms of guiding the choice of anti-TOI (VEGF-A, PDGF-B and/or PDGFR-B) ligand for administration to human patients for therapy and/or prophylaxis of VEGF-A, PDGF-B and/or PDGFR-B-mediated or associated diseases or conditions. In this way, the patient receives drugs and ligands that are tailored to their needs—as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

In developing this thinking, in a non-limiting embodiment the present inventor decided to determine a set of human VEGF-A, PDGF-B and PDGFR-B variants on the basis of the following criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population. The inventor selected variants having at least 3 of the 4 following criteria:—

Naturally-occurring human TOI (VEGF-A, PDGF-B or PDGFR-B) variation having a cumulative human allele frequency of 35% or less;

Naturally-occurring human TOI variation having a total human genotype frequency of about 50% or less;

Naturally-occurring human TOI variation found in many different human ethnic populations (using the standard categorisation of the 1000 Genomes Project; see Table 2 below); and

Naturally-occurring human TOI variation found in many individuals distributed across such many different ethnic populations.

On the basis of these criteria, the inventor identified variants listed below.

Furthermore, the inventor identified useful variations also in the following human TOIs: complement factor H (CFH), complement component 2 (C2), prothrombin, methylenetetrahydrofolate reductase (MTHFR), Factor V, ARMS2, VEGFR2 and HTRA1. As the titles to Tables 18-20 indicate, the examiner realised that such variants have useful correlations.

In an example, the ligand of the invention comprises an anti-TOI (eg, anti-human VEGF-A) binding site, wherein the binding site is a human or humanized binding site, eg, the binding site comprises or consists of a human or humanized antibody variable domain or plurality of variable domains (eg, human VH/VL binding site(s)). Additionally or alternatively, the ligand comprises one or more human antibody constant regions (eg, a human antibody CH1, CH2, CH3 (or all of these) or Fc). In an example, the ligand is an antibody that comprises human or humanized variable regions and human constant regions (eg, bearing one or more mutations to enhance or dampen Fc function in a human patient).

In an example, the invention provides a method of targeting VEGF-A in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human VEGF-A protein that is encoded by a human VEGF-A nucleotide comprising a VEGF-A SNP as described herein. In an example, the human is suffering from or at risk of a VEGF-A-mediated disease or condition. In an example, the method treats or reduces the risk of a VEGF-A-mediated disease or condition in the human. In an example, the condition comprises angiogenesis or neovascularisation.

In an example, the invention provides a method of targeting PDGF-B in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human PDGF-B protein that is encoded by a human PDGF-B nucleotide comprising a PDGF-B SNP as described herein. In an example, the human is suffering from or at risk of a PDGF-B-mediated disease or condition. In an example, the method treats or reduces the risk of a PDGF-B-mediated disease or condition in the human. In an example, the condition comprises angiogenesis or neovascularisation.

In an example, the invention provides a method of targeting PDGFR-B in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human PDGFR-B protein that is encoded by a human PDGFR-B nucleotide comprising a PDGFR-B SNP as described herein. In an example, the human is suffering from or at risk of a PDGFR-B-mediated disease or condition. In an example, the method treats or reduces the risk of a PDGFR-B-mediated disease or condition in the human. In an example, the condition comprises angiogenesis or neovascularisation.

In an embodiment, (i) the antibody or fragment comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and wherein (ii) said human comprises said nucleotide sequence encoding said VEGF-A, PDGF-B or PDGFR-B.

Additionally or alternatively, in an embodiment, (i) the antibody or fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said human comprises said nucleotide sequence encoding said VEGF-A, PDGF-B or PDGFR-B.

In a specific embodiment, the anti-VEGF-A, PDGF-B or PDGFR-B ligand, antibody or fragment of present invention comprises an Fc region, wherein the Fc region comprises at least one non-native amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 33IT, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may comprise additional and/or alternative non-native amino acid residues known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

The ligand, antibody or fragment according to the invention (eg, an anti-VEGF-A ligand, antibody or fragment) is for treating or preventing or reducing the risk of (or treats or prevents or reduces the risk of), for example, any disease or condition disclosed in US2012064621A1 or U.S. Pat. No. 7,704,500B2, the disclosure of which diseases and conditions are incorporated herein by reference for potential inclusion in one or more claims herein. Guidance on obtaining and testing antibodies can also be found in that PCT application. Suitable anti-VEGF-A ligands, antibodies or fragments for use in the invention are disclosed in US2012064621A1 or U.S. Pat. No. 7,704,500B2, the disclosure of which (including sequences thereof) are incorporated herein by reference for potential inclusion in one or more claims herein.

Further encompassed by the invention is the use of the anti-TOI (eg, VEGF-A) ligand, antibody or fragment in the manufacture of a medicament for use to attenuate or inhibit a TOI (eg, VEGF-A)-mediated disease or disorder in a human. VEGF-A-mediated or related disorders which are treated by the ligand, antibody or fragment of the invention include, for example, described below.

Thus, a ligand, antibody or fragment of the invention is useful as a therapeutic agent in the treatment of a condition involving TOI (eg, VEGF-A) expression and/or activity. One embodiment, among others, is a method of treatment comprising administering an effective amount of a ligand, antibody or fragment of the invention to a patient in need thereof, wherein functional consequences of TOI activation are decreased. Another embodiment, among others, is a method of treatment comprising (i) identifying a patient demonstrating TOI expression or activity, and (ii) administering an effective amount of a ligand, antibody or fragment of the invention to the patient, wherein a functional consequence of TOI activation are attenuated. An effective amount according to the invention is an amount that modulates (e.g. decreases) the functional consequences of TOI activation so as to modulate (e.g. decrease or lessen) the severity of at least one symptom of the particular disease or disorder being treated, but not necessarily cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, comprising administering to a patient in need thereof an effective amount of one or more ligands, antibodies or fragments of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein such that the severity of at least one symptom of any of the disorders is reduced. Another embodiment of the invention, among others, is a method of antagonizing at least one effect of TOI comprising contacting with or administering an effective amount of one or more ligands, antibodies or fragments of the present invention such that said at least one effect of TOI is antagonized, e.g. the ability to promote or maintain angiogenesis or neovascularisation.

Tailoring Antibodies to Rarer TOI (VEGF-A, PDGF-B or PDGFR-B) Variant Profile

As outlined herein (for example, in the context of PCSK9 in Example 1), the invention includes the possibility to tailor treatment of humans further by selecting antibody-based ligands with variable domains and/or constant domains based on gene segments found in many humans of the ethnic populations where the variant TOI forms are found to meet the selection criteria of the invention. This also applies mutatis mutandis where the TOI is human VEGF-A, PDGF-B or PDGFR-B as in the present example. Thus, all disclosure herein relating to tailoring variable and/or constant domains apply to the present example, relating to VEGF-A, PDGF-B or PDGFR-B and is combinable for use in one or more claims herein.

As described in Example 1, an example is provided for ligands comprising antibody VH domains derived from recombination of human IGHV gene segments comprising selected nucleotides at positions in the HCDR1 or FW3 where there is variability in humans (ie, where SNPs occur in humans).

Further information is provided in Table 4, which shows variation at these positions, as well as the variant distributions across the 1000 Genomes Project database relating to many human populations.

In other embodiments, as explained more fully above, the invention provides for ligands which are tailored to the human recipient's genotype and/or phenotype based on alternative human VH gene segments, or on Vκ, Vλ or constant region gene segments (see further Table 9 for representative variants).

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23*04), a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40. (ii) wherein the ligand comprises a VH domain derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment, and wherein said human comprises a IGHV3-7*01 VH gene segment or the human expresses VH domains derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment. (iii) wherein the ligand comprises a Vκ domain derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment, and wherein said human comprises a IGKV1-12*01 Vκ gene segment or the human expresses Vκ domains derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment. (iv) wherein the ligand comprises a Vκ domain derived from the recombination of a human Vκ segment and a human Jκ segment, the human Vκ segment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36 and wherein said human comprises a Vκ gene segment encoding a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the human expresses Vκ domains that comprise a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 and wherein said human comprises a Vκ gene segment encoding a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 or the human expresses Vκ domains that comprise a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38. (v) wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4. (vi) wherein the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6. (vii) wherein the ligand comprises a human kappa chain constant region that comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16 and wherein said human comprises (i) an IGKC1*01 human kappa chain constant region gene segment, or the human expresses antibodies comprising human kappa chain constant regions comprising a Val corresponding to position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16. (viii) wherein the ligand comprises a human IGLC1*01 lambda chain constant region and wherein said human comprises (i) a human IGLC1*01 lambda chain constant region gene segment, or the human expresses antibodies comprising human IGLC1*01 lambda chain constant regions. (ix) wherein the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73. (x) wherein the ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3*01) constant region gene segment and wherein said human comprises (i) said first constant region gene segment (eg, an IGHG3*01), or the human expresses antibodies comprising human gamma-3 heavy chain constant regions encoded by said first human IGHG3 (eg, IGHG3*01) constant region gene segment. (xi) wherein the ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human epsilon heavy chain constant regions encoded by said first constant region gene segment. (xii) wherein the ligand comprises a human mu heavy chain constant region encoded by a first human mu heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human mu heavy chain constant regions encoded by said first constant region gene segment. (xiii) wherein the ligand comprises a human alpha heavy chain constant region encoded by a first human alpha heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human alpha heavy chain constant regions encoded by said first constant region gene segment. (xiv) wherein the ligand comprises a human delta heavy chain constant region encoded by a first human delta heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human delta heavy chain constant regions encoded by said first constant region gene segment. (xv) wherein the ligand comprises a human kappa light chain constant region encoded by a first human kappa light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human kappa light chain constant regions encoded by said first constant region gene segment. (xvi) wherein the ligand comprises a human lambda light chain constant region encoded by a first human lambda light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human lambda light chain constant regions encoded by said first constant region gene segment.

It may be advantageous for VEGF-A, PDGF-B or PDGFR-B ligands, for the ligand to comprise a gamma-1 or gamma-2 constant region (eg, as per the embodiments (i) to (xvi) above). For example, the ligand comprises said gamma-1 constant region, eg, an ADCC or CDC enhanced constant region meeting embodiment (v).

Determination of Specific Binding of Ligands of the Invention to VEGF-A, PDGF-B or PDGFR-B Variants

The specific binding of ligands of the invention to VEGF-A, PDGF-B or PDGFR-B variants can be performed using the SPR method described in Example 1.

Human Polymorphisms

REFERENCES

-   1. PLoS One. 2013 Dec. 20; 8(12):e84069. doi:     10.1371/journal.pone.0084069, eCollection 2013, “Association of VEGF     gene polymorphisms with diabetic retinopathy: a meta-analysis”, Gong     J Y et al. The authors reported a meta analysis which, in     conclusion, reportedly demonstrated that DR is associated with VEGF     gene −460T C polymorphism (rs833061). -   2. J Diabetes Res. 2014; 2014:805801. doi: 10.1155/2014/805801, Epub     2014 Apr. 28, “The associations between VEGF gene polymorphisms and     diabetic retinopathy susceptibility: a meta-analysis of 11     case-control studies”, Han L et al. A total of 11 studies fulfilling     the inclusion criteria were included in this meta-analysis. A     significant relationship between VEGF+936C/T (rs3025039)     polymorphism and DR was reported. -   3. BMC Ophthalmol. 2013 Oct. 16; 13:56. doi:     10.1186/1471-2415-13-56, “Two polymorphisms (rs699947, rs2010963) in     the VEGF-A gene and diabetic retinopathy: an updated meta-analysis”,     Lu Y et al. The authors reported meta-analysis confirmed the     significant association between rs699947 polymorphism and DR after     exclusion of outliers. -   4. Gene. 2013 Apr. 15; 518(2):310-5. doi:     10.1016/j.gene.2013.01.018. Epub 2013 Jan. 24. VEGF −634G>C     polymorphism and diabetic retinopathy risk: a meta-analysis. Qiu M     et al. The aim of the published study was to investigate whether     VEGF −634G>C polymorphism is associated with the risk of DR in type     2 diabetes. A systematic search of electronic databases (PubMed,     Embase and Web of Science) and reference lists of relevant articles     was carried out until Sep. 15, 2012. The pooled odds ratios (ORs)     and their corresponding 95% confidence interval (CI) were calculated     by a fixed effect model. A total of 1525 DR cases and 1422 diabetic     without retinopathy (DWR) controls in 9 independent studies were     included in the meta-analysis. A significant relationship between     VEGF −634G>C polymorphism and DR was found in an allelic genetic     model (OR: 1.13, 95% CI: 1.01 to 1.25, P=0.03) and a recessive     genetic model (OR: 1.26, 95% CI: 1.02 to 1.55, P=0.03). The authors     concluded that their research confirmed the association between the     VEGF −634G>C polymorphism and DR in subjects with type 2 diabetes. -   5. Diabetes. 2002 May; 51(5):1635-9, “A common polymorphism in the     5′-untranslated region of the VEGF gene is associated with diabetic     retinopathy in type 2 diabetes”, Awata T et al. These authors     suggest that the −634G>C polymorphism in the 5′UTR of the VEGF gene     is a genetic risk factor for diabetic retinopathy. -   6. Molecular Vision 2010;     16:1958-1981<http://www.molvis.org/molvis/v16/a213>, Tong Y et al.     In conclusion, according to the authors the described Human Genome     Epidemiology (HuGE) systematic review presents strong evidence for     an association between the HTRA1 rs11200638 G→A polymorphism,     LOC387715/ARMS2rs10490924 G-T polymorphism, and AMD, and suggests     that both of these genes play important roles in this disease.     Potential gene-gene and gene-environmental interactions and possible     mechanisms of AMD are also summarized and discussed. -   7. J. Pers. Med. 2013, 3, 40-69; doi:10.3390/jpm3010040,     “Personalized Medicine in Ophthalmology: From Pharmacogenetic     Biomarkers to Therapeutic and Dosage Optimization”, Frank S Ong et     al. This review comments that: In the case of intravitreal     bevacizumab, CFH Y402H genotypes, TC and TT, show more than     five-fold increased improvement compared to the CC genotype. The     data shows that after treatment with bevacizumab, visual acuity of     the patients improved from 20/248 to 20/166 (TT) and from 20/206 to     20/170 (TC), but actually decreased from 20/206 to 20/341 for the CC     genotype (p=0.016). In a prospective study with twice the number of     patients, the CC genotype was confirmed to have worse outcome as     measured by distance and reading visual acuity. In a similar     experiment with intravitreal ranibizumab, the TC and TT genotypes     for CFH showed improvement with fewer injections compared to the CC     genotype. Recurrent analysis showed that patients homozygous for the     CFH Y402H risk allele (CC) were 37% more likely to require     additional ranibizumab injections (p=0.04). Another study found that     individuals homozygous for 69S in ARMS2 had decreased central     subfield retinal thickness and no improvement in visual outcomes     compared to improved visual acuity in ARMS2 rs10490924 and     rs11061170 genotypes following ranibizumab treatment. Taking     genetics and environmental factors together, the CFH Y402H     homozygous CC genotype with BMI ≥30 and smoking reportedly conferred     the greatest risk. -   8. Expert Rev Ophthalmol. 2013 April 1; 8(2): 127-140.     doi:10.1586/eop.13.3, “Genetic risk, ethnic variations and     pharmacogenetic biomarkers in age-related macular degeneration and     polypoidal choroidal vasculopathy”, Jane Z. Kuo et al. This article     comments that: Variants in the complement factor H (CFH) gene     confers a 7.4-fold increased likelihood of late AMD. AMD is a common     complex disorder with complex inheritance patterns due to various     gene and environmental interactions. Yet, despite these challenges,     AMD represents an unusual example of the “common disease common     variant” theory in which several variants identified on the CFH gene     have relatively large effects on disease outcome. Even though the     most well-established and consistent association of AMD is observed     with complement factor H (CFH) on chromosome 1, recent studies have     also shown that other candidate genes in the complement pathway,     ARMS2/HTRA1 on chromosome 10q26, LIPC, CETP, and TIMP2 are also     susceptible loci associated with AMD. In 2005, three independent     research groups identified a common coding variant, Y402H     (rs1061170), in the CFH gene on chromosome 1 that was strongly     associated with susceptibility of AMD, with reported odds ratio (OR)     ranging from 2.5 to 3.3 for all AMD and as high as 7.4 for advanced     AMD. The Y402H variant results in a tyrosine-to-histidine     substitution. The association of CFH in AMD implicated that the     complement cascade plays a critical role in the development of AMD     and is now also regarded as a major contributor to susceptibility of     AMD. In addition to CFH and the complement pathway, the region at     chromosome 10q26 also harbors additional signals to AMD     susceptibility, namely the age-related maculopathy susceptibility 2     (ARMS2) and the high-temperature requirement factor H (HTRA1) genes.     It is also possible that susceptible variants within ARMS2 modulate     the promoter activity of HTRA1, so that both ARMS2/HTRA1 contribute     to AMD susceptibility. In one European study, the SNP rs11200638 at     the promoter region of HTRA1 confers a 49.3% attributable risk and     the risk allele was associated with an elevated mRNA and protein     expression and level of HTRA1. The risk allele of the same SNP was     highly significant in the Chinese and has 10 times the risk of     developing AMD compared to subjects with the wild-type allele. Two     VEGF polymorphisms, rs699947 and rs2146323, were significantly     associated with PDT treatment response. The allele frequency of     rs699947 for AA, AC, and CC were 14%, 39%, and 46% in non-responders     versus 40%, 48%, and 12% in responders, respectively (p=0.0008). The     allele frequency of rs2146323 for AA, AC, and CC were 4%, 32%, and     64% in non-responders versus 24%, 38%, and 38% in responders,     respectively (p=0.0369). Thus, the C allele in both SNPs in VEGF     were associated with a higher rate of non-responder to PDT (p=0.0003     for rs699947 and p=0.0036 for rs2146323). In classic choroidal     neovascularization (CNV), prothrombin (G20210A) and MTHFR (C677A)     were significantly associated with PDT responders. In occult CNV,     prothrombin (G20210A) and factor V (G1691A) were significantly     associated with PDT responders. Two studies have showed a worse     visual improvement in subjects with the homozygous TT risk allele at     ARMS2 rs10490924 (A69S) after ranibizumab or bevacizumab injections.     Other candidate gene studies found that patients with the risk     genotype (CC) at rs1413711 of VEGF has a more significant visual     gain after ranibizumab, the G allele at rs699946 of VEGF was     significantly associated with a better visual prognosis after either     intravitreal bevacizumab or triple therapy (PDT with intravitreal     bevacizumab and triamcinolone acetonide). In a study examining the     cumulative effect of risk alleles at CFH (Y402H; rs1061170), ARMS2     (A69S; rs10490924), VEGF (rs699947 and rs833069), KDR (rs2071559 and     rs7671745), LPR5 (rs3736228), and FZD4 (rs10898563) after     ranibizumab treatment, subjects without the high-risk alleles in CFH     and ARMS2 have significantly more visual improvement compared to     subjects with the high-risk alleles (p=0.009). The mean visual     acuity improvement was 10 letters in subjects without the 4-risk     alleles, compared to no improvement in subjects with all 4-risk     alleles. By adding VEGF into the model, they found that subjects     with all 6-risk alleles in CFH, ARMS2, and VEGF demonstrated a mean     loss of 10 letters after ranibizumab treatment, whereas the other     allele groups all demonstrated improvement (p<0.0001), suggesting a     cumulative effect of the risk alleles with a poorer response rate to     treatment. A comprehensive meta-analysis found variants at     ARMS2/HTRA1 (rs10490924, OR=2.27, p<0.00001; rs11200638, OR=2.72,     p<0.00001), CFH (rs1061170, OR=1.72, p<0.00001; rs800292, OR=2.10,     p<0.00001) or the complement pathway (C2; rs547154, OR=0.56, p=0.01)     were significantly associated with PCV, with higher odds ratio     observed at variants in ARMS2/HTRA1. The strong association of     ARMS2/HTRA1 to PCV was supported by numerous studies of Asian     populations. -   9. Survey of Ophthalmology, Volume 59, Issue 1, January-February     2014, Pages 1-18, “Predictors of anti-VEGF treatment response in     neovascular age-related macular degeneration”, Finger et al. The     authors reviewed evidence on predictors of anti-vascular endothelial     growth factor (VEGF) treatment response in neovascular age-related     macular degeneration. Just under half of the SNPs assessed in the     CFH gene and 15% of the SNPs assessed in the VEGF gene were found to     be associated with visual outcomes or the number of injections     required during follow-up. -   10. Ophthalmology. 2013 January; 120(1):115-21. doi:     10.1016/j.ophtha.2012.10.006. Epub 2012 Nov. 11, “Variants in the     VEGFA gene and treatment outcome after anti-VEGF treatment for     neovascular age-related macular degeneration2, Abedi F et al. The     authors concluded that: Pharmacogenetic association with anti-VEGF     treatments may influence the visual outcomes in neovascular AMD. In     patients with the T allele in tSNP rs3025000, there was a     significantly better visual outcome at 6 months and a greater chance     of the patients belonging to the responder group with anti-VEGF     treatment at 3, 6, and 12 months. The visual acuity (VA) outcomes of     patients harbouring the T allele at SNP rs3025000 were comparable     with those of the pivotal clinical trials but with fewer injections. -   11. Ophthalmology. 2014 April; 121(4):905-10. doi:     10.1016/j.ophtha.2013.10.047. Epub 2013 Dec. 21, “Polymorphisms in     vascular endothelial growth factor receptor 2 are associated with     better response rates to ranibizumab treatment in age-related     macular degeneration”, Hermann M M et al. The authors evaluated the     association of single nucleotide polymorphisms (SNPs) in VEGF genes     and their receptors (VEGFR) with the response rate to ranibizumab in     366 patients with neovascular AMD. Univariate analyses of variance     (ANOVAs) revealed a significant effect of SNP rs4576072 in the     VEGFR2 gene on VA change after 12 months (F[1,235]=14.05; P=0.02). A     stepwise linear regression analysis returned a model (P=0.01) with     SNPs rs4576072 and rs6828477 in the VEGFR2 gene as independent     predictors for VA change after 12 months, with a mean increase in VA     of 0.26 on the logarithm of the minimum angle of resolution (log     MAR) scale in patients with 3 contributing minor alleles compared     with a loss of 0.03 log MAR in patients with no minor allele. -   12. Research and Reports in Neonatology 2011:1 5-11; “VEGF 936C>T is     predictive of threshold retinopathy of prematurity in Japanese     infants with a 30-week gestational age or less”, Yagi M et al. The     authors found that VEGF 936C>T polymorphism and duration of oxygen     administration were independent risk factors for threshold ROP using     a multivariate logistic analysis with adjustment for gestational age     and birth weight. -   13. Clin Cancer Res 2009; 15(17) Sep. 1, 2009, pp 5297-5302, DOI:     10.1158/1078-0432.CCR-08-2576, “The Role of Vascular Endothelial     Growth Factor Genetic Variability in Cancer”, Bryan P. Schneider et     al. The authors comment that recently, genetic variability in VEGF     has been studied as a potential predictive biomarker for     bevacizumab. The VEGF-1154 AA and -2578 AA genotypes predicted an     improved median overall survival, whereas the VEGF-634 CC and -1498     TT genotypes predicted protection from grade 3-4 hypertension in the     pivotal trial, E2100. If validated, these finding could help direct     which subgroup of patients should receive bevacizumab.

The inventor identified the following SNPs as SNPs of interest for the present invention.

TABLE 18 POLYMORPHISM RISK FACTORS FOR OCULAR CONDITIONS SNP LOCATION ASSOCIATED (forward EXAMPLE POLYMORPHISM SNP ID strand) INDICATION VEGF-A −2578C > A rs699947 6: 43768652 DR −460T > C rs833061 6: 43769749 DR −634G > C rs2010963 6: 43770613 DR +936C > T rs3025039 6: 43784799 DR ROP A > G rs699946 6: 43764932 DR HTRA1 −625G > A rs11200638 10: 122461028 AMD ARMS2 G > T rs10490924 10: 122454932 AMD A69S PCV COMPLEMENT FACTOR H (CFH) 184G > A rs800292  1: 196673103 AMD V62I PCV COMPLEMENT COMPONENT 2 (C2) 62G > T rs547154 6: 31943161 AMD PCV NB: as is known in the art SNP location is expressed as chromosome number: nucleotide position, eg, 6: 43768652 is nucleotide number 43768652 on human chromosome 6. ARMS2 is also known as age-related maculopathy susceptibility protein 2, ARMD8 and ARMS2_HUMAN LOC387715.

TABLE 19 POLYMORPHISMS ASSOCIATED WITH IMPROVED OCULAR RESPONSES SNP LOCATION (forward ASSOCIATED EXAMPLE POLYMORPHISM SNP ID strand) BENEFIT VEGF-A −2578C > A rs699947 6: 43768652 AA and AC genotypes associated with improved PDT response −111C > A rs2146323 6: 43777358 AA and AC genotypes associated with improved PDT response −1400T > C rs1413711 6: 43772941 CC genotype shows more significant visual gain after anti- VEGF treatment A > G rs699946 6: 43764932 G significantly associated with better visual prognosis after anti- VEGF treatment 398G > A rs833068 6: 43774790 AA or GG genotype associated with less anti-VEGF treatment injections needed 450T > C rs833069 6: 43774842 AA (TT) or GG (CC) genotype associated with less anti-VEGF treatment injections needed −28C > T rs3025000 6: 43778432 T allele associated with visual acuity improvement with lower anti-VEGF administrations COMPLEMENT FACTOR H (CFH) T > C rs1061170  1: 196690107 TC and TT genotypes: Improved Y402H visual acuity following anti- VEGF treatment −2362T > C rs3766404  1: 196682702 TT (AA) genotype associated with less anti-VEGF treatment injections needed prothrombin 20210G > A rs1799963 11: 46739505  Associated with improved PDT response Methylenetetrahydrofolate reductase (MTHFR) 677C > A rs1801133 1: 11796321 Associated with improved PDT A222V response FACTOR V 1691G > A rs6025  1: 169549811 Associated with improved PDT R506Q response ARMS2 G > T rs10490924 10: 122454932 GG genotype associated with A69S improved anti-VEGF response VEGFR2 1026A > G rs4576072 4: 55120071 G allele associated with better visual acuity following anti- VEGF 1263G > A rs6828477 4: 55100634 A allele associated with better visual acuity following anti- VEGF

TABLE 20 POLYMORPHISMS ASSOCIATED WITH IMPROVED CANCER RESPONSES SNP LOCATION ASSOCIATED (forward EXAMPLE POLYMORPHISM SNP ID strand) BENEFIT VEGF-A −2578C > A rs699947 6: 43768652 AA genotype associated with improved breast cancer response to anti-VEGF −1154G > A rs1570360 6: 43770093 AA genotype associated with improved breast cancer response to anti-VEGF

The invention thus provides the following concepts:—

Human VEGF-A SNPs Associated With VEGF-A Mediated Diseases & Conditions

1. A method of treating or reducing the risk of a disease or condition mediated by VEGF-A in a human, the method comprising administering to said human an anti-VEGF-A ligand (eg, an anti-VEGF-A trap, antibody or antibody fragment) that specifically binds to a human VEGF-A that is expressed by a VEGF-A nucleotide sequence comprising a SNP selected from the group consisting of rs699947, rs833061, rs2010963, rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069, rs3025000 and rs1570360, wherein said human comprises a VEGF-A nucleotide sequence comprising said selected SNP.

Herein, specific binding can be tested using a sample VEGF-A using a routine method known the skilled person, eg, ELISA or SPR (eg, using a method as described herein). For example, the VEGF-A is provided by a sample from said human or another human, eg, a serum or blood sample.

In an example, the ligand comprises an antibody constant region (eg, an antibody Fc region). Optionally, the ligand comprises a human gamma-1 heavy chain constant region that comprises an amino acid selected from the group consisting of an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising said selected amino acid. Optionally, this constant region is comprised by an Fc region of the ligand.

In an example, the ligand comprises an anti-VEGF-A binding site that specifically binds human VEGF-A. Optionally, the binding site comprises one or more antibody variable domains or one or more human VEGF-A receptor domains. In an example, specific binding with a Kd of 1 mm or less (ie, 1 mM or stronger binding), eg, 1 nM or less; or 100 pM or less; or 10 pM or less. Specific binding can be determined, for example, by assessing binding of the ligand to VEGF-A in a sample from said human or another human comprising said VEGF-A. In an example, the sample comprises serum, blood, faeces, tissue, a cell, urine and/or saliva of said human.

Optionally, the VEGF-A is a VEGF-A-165 isoform.

In an example, the ligand is an anti-VEGF-A trap, eg, aflibercept. In an example, the ligand is EYLEA™.

In an example, the ligand is an anti-VEGF-A antibody or antibody fragment, eg, ESBA1008, bevacizumab or ranizumab. In an example, the ligand is AVASTIN™ or LUCENTIS™.

In an example, the ligand is an anti-VEGF ankyrin repeat protein (DARPin), eg, abicipar pegol.

In an example, the ligand is an NCE (so-called New Chemical Entity in the art), eg, sunitinib.

In an example, the condition is selected from the group consisting of angiogenesis, neovascularisation, ocular vascularisation and solid tumour vascularisation.

In an example, the condition is subretinal neovascularisation.

In an example, the disease or condition is an angiogenic disease or condition.

Optionally the disease or condition is an angiogenic ocular condition or angiogenic cancer, eg a solid tumour, a gastrointestinal cancer, colorectal cancer, liver cancer, breast cancer, ovarian cancer, non small cell lung cancer, thyroid cancer, an oral cancer, or an haematological cancer.

Optionally the angiogenic disease or condition is selected from the group consisting of psoriasis, rheumatoid arthritis, a hemangioma, an angiofibroma, diabetic retinopathy, corneal neovacularisation and neovascular glaucoma.

Optionally the disease or condition is or comprises vascular permeability (eg, ocular vascular permeability), edema or inflammation in said human.

For example, the disease or condition is selected from the group consisting of brain edema (eg, edema associated with brain injury, stroke or brain tumour); edema associated with an inflammatory disorder (eg, associated with psoriasis or arthritis, rheumatoid arthritis or asthma); edema associated with burns; ascites or pleural effusion (eg, associated with tumours, inflammation or trauma); chronic airway inflammation; capillary leak syndrome; sepsis; kidney disease (eg, associated with increased leakage of protein); and an eye disorder (eg, age related macular degeneration or diabetic retinopathy).

In an example the method is for reducing or reducing the risk of VEGF-A mediated endothelial cell maintenance or proliferation in an angiogenic disease or condition in said human.

In an example, the angiogenic disease or condition is an angiogenic disease or condition recited above, eg, an angiogenic ocular condition.

In an example the method is for regressing neovascularisation in a VEGF-A mediated disease or condition in said human.

In an example the method is for treating or reducing the risk of neovascularisation in a human, wherein the human has VEGF-A ligand-refractory neovascularisation.

In an example, the human has been diagnosed with said disease or condition before said administration of the ligand.

For example, the human is partially or completely resistant to treatment with an anti-VEGF-A ligand, eg, aflibercept, sunitinib, bevacizumab or ranizumab.

In an example, the ligand is administered by intravenous or subcutaneous administration and/or is comprised in an injectable preparation.

2. The method of concept 1, wherein the condition is an ocular condition, eg, diabetic retinopathy or retinopathy of prematurity.

In an example, the condition is diabetic retinopathy (DR).

In an example, the condition is age-related macular degeneration (AMD), eg, exudative AMD.

In an example, the condition is polypoidal choroidal vasculopathy (PCV).

In an example, the condition is retinal angiomatous proliferation (RAP).

In an example, the condition is diabetic macular edema (DME).

In an example, the condition is retinal-vein occlusion (RVO).

In an example, the condition is retinopathy of prematurity (ROP).

In an alternative, instead of “a method of treating or reducing the risk of a disease or condition mediated by VEGF-A in a human”, the invention relates to “a method of improving visual acuity in a human”.

3. The method of concept 1 or 2, wherein the SNP is selected from the group consisting of rs699947, rs833061, rs2010963, rs3025039 and rs699946.

In an example, the SNP is rs699947 and optionally the condition is DR.

In an example, the SNP is rs833061 and optionally the condition is DR.

In an example, the SNP is rs2010963 and optionally the condition is DR.

In an example, the SNP is rs3025039 and optionally the condition is DR.

In an example, the SNP is rs699946 and optionally the condition is DR.

4. The method of concept 1 or 2, wherein the SNP is selected from the group consisting of rs699947, rs2146323, rs1413711, rs699946, rs833068, rs833069 and rs3025000.

As shown in Table 19, these SNPs are associated with improved ocular responses following ocular treatment (eg, photodynamic therapy (PDT) or VEGF-A antagonism).

In an example, the SNP is rs699947, eg, homozygous for said SNP.

In an example, the SNP is rs2146323, eg, homozygous for said SNP.

In an example, the SNP is rs1413711, eg, homozygous for said SNP.

In an example, the SNP is rs699946, eg, homozygous for said SNP.

In an example, the SNP is rs833068, eg, homozygous for said SNP.

In an example, the SNP is rs833069, eg, homozygous for said SNP.

In an example, the SNP is rs3025000, eg, homozygous for said SNP.

5. The method of concept 3 or 4, wherein the human is homozygous for said SNP.

In another example, the human is heterozygous for said SNP.

6. The method of any one of concepts 2 to 5, wherein the method further comprises treating the human with photodynamic therapy.

For example, the method further comprises treating the human with PDT simultaneously with, or after, administration of said ligand to the human.

7. The method of concept 1, wherein the disease or condition is a cancer. 8. The method of concept 7, wherein the SNP is rs699947 or rs1570360.

In an example, the SNP is rs699947, eg, homozygous for said SNP.

In an example, the SNP is rs1570360, eg, homozygous for said SNP.

9. The method of any preceding concept, wherein the human comprises (a) a HTRA1 nucleotide sequence comprising SNP rs11200638; (b) an ARMS2 nucleotide sequence comprising SNP rs10490924; (c) a CFH nucleotide sequence comprising SNP rs800292, a T at the position of rs1061170 or an A at the position of rs3766404; (d) a complement component 2 (C2) nucleotide sequence comprising SNP rs547154; (e) a prothrombin nucleotide sequence comprising SNP rs1799963; (f) a MTHFR nucleotide sequence comprising SNP rs1801133; (g) a Factor V nucleotide sequence comprising SNP rs6025; or (h) a VEGFR2 nucleotide sequence comprising SNP rs4576072 or rs6828477.

In an example the human comprises a HTRA1 nucleotide sequence comprising SNP rs11200638, eg, in homozygous form.

In an example the human comprises an ARMS2 nucleotide sequence comprising SNP rs10490924, eg, in homozygous form.

In an example the human comprises a CFH nucleotide sequence comprising SNP rs800292, eg, in homozygous form.

In an example the human comprises a CFH nucleotide sequence comprising SNP rs1061170, eg, in homozygous form.

In an example the human comprises a CFH nucleotide sequence comprising SNP rs3766404, eg, in homozygous form.

In an example the human comprises a complement component 2 (C2) nucleotide sequence comprising SNP rs547154, eg, in homozygous form.

In an example the human comprises a prothrombin nucleotide sequence comprising SNP rs1799963, eg, in homozygous form.

In an example the human comprises a MTHFR nucleotide sequence comprising SNP rs1801133, eg, in homozygous form.

In an example the human comprises a Factor V nucleotide sequence comprising SNP rs6025, eg, in homozygous form.

In an example the human comprises a VEGFR2 nucleotide sequence comprising SNP rs4576072, eg, in homozygous form.

In an example the human comprises a VEGFR2 nucleotide sequence comprising SNP rs6828477, eg, in homozygous form.

10. The method of any one of concepts 2 to 6, wherein the human comprises (a) a HTRA1 nucleotide sequence comprising SNP rs11200638; (b) an ARMS2 nucleotide sequence comprising SNP rs10490924; (c) a CFH nucleotide sequence comprising SNP rs800292; or (d) a complement component 2 (C2) nucleotide sequence comprising SNP rs547154.

These SNPs are risk factors for ocular conditions.

In an example, the ocular condition is DR or ROP and the method is for treating or reducing the risk of DR or ROP.

11. The method of any one of concepts 2 to 6, wherein the human comprises (i) an ARMS2 nucleotide sequence comprising a G at the position of SNP rs10490924; (ii) a CFH nucleotide sequence comprising a T at the position of SNP rs1061170 or rs3766404; (iii) a prothrombin nucleotide sequence comprising SNP rs1799963; (iv) a MTHFR nucleotide sequence comprising SNP rs1801133; (v) a Factor V nucleotide sequence comprising SNP rs6025; or (vi) a VEGFR2 nucleotide sequence comprising SNP rs4576072 or rs6828477.

These SNPs are associated with improved responses following treatment (eg, PDT and/or VEGF-A antagonism) of an ocular disease or condition.

In an example, the ocular condition is DR and the method is for treating or reducing the risk of DR.

SNPs Associated With Improved Responses Following Anti-VEGF-A Treatment of An Ocular Disease Or Condition

12. A method of treating or reducing the risk of a disease or condition mediated by VEGF-A in a human, the method comprising administering to said human an anti-VEGF-A ligand (eg, an anti-VEGF-A trap, antibody or antibody fragment) that specifically binds to a human VEGF-A, wherein the human comprises (i) an ARMS2 nucleotide sequence comprising a G at the position of SNP rs10490924; (ii) a CFH nucleotide sequence comprising a T at the position of SNP rs1061170 or an T at the position of rs3766404; or (iii) a VEGFR2 nucleotide sequence comprising SNP rs4576072 or rs6828477, wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an amino acid selected from the group consisting of an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising said selected amino acid.

These SNPs are associated with improved responses following anti-VEGF-A treatment of an ocular disease or condition.

In an example, the ocular condition is DR and the method is for treating or reducing the risk of DR. In an example the human comprises an ARMS2 nucleotide sequence comprising G at the position of SNP rs10490924, eg, in homozygous form.

In an example the human comprises a CFH nucleotide sequence comprising a T at the position of SNP rs1061170, eg, in homozygous form.

In an example the human comprises a CFH nucleotide sequence comprising a T at the position of SNP rs3766404, eg, in homozygous form.

In an example the human comprises a VEGFR2 nucleotide sequence comprising SNP rs4576072, eg, in homozygous form.

In an example the human comprises a VEGFR2 nucleotide sequence comprising SNP rs6828477, eg, in homozygous form.

13. The method of concept 12, wherein the condition is an ocular condition, eg, diabetic retinopathy or retinopathy of prematurity.

In an example, the condition is diabetic retinopathy (DR).

In an example, the condition is age-related macular degeneration (AMD), eg, exudative AMD.

In an example, the condition is polypoidal choroidal vasculopathy (PCV).

In an example, the condition is retinal angiomatous proliferation (RAP).

In an example, the condition is diabetic macular edema (DME).

In an example, the condition is retinal-vein occlusion (RVO).

In an example, the condition is retinopathy of prematurity (ROP).

In an alternative, instead of “a method of treating or reducing the risk of a disease or condition mediated by VEGF-A in a human”, the invention relates to “a method of improving visual acuity in a human”.

14. The method of concept 12 or 13, wherein the human further comprises a VEGF-A SNP selected from the group consisting of rs699947, rs833061, rs2010963, rs3025039 and rs699946.

In an example, the SNP is rs699947.

In an example, the SNP is rs833061.

In an example, the SNP is rs2010963.

In an example, the SNP is rs3025039.

In an example, the SNP is rs699946.

15. The method of any one of concepts 12 to 14, wherein the method further comprises treating the human with photodynamic therapy.

For example, the method further comprises treating the human with PDT simultaneously with, or after, administration of said ligand to the human.

16. The method of concept 12, wherein the disease or condition is a cancer. 17. The method of concept 16, wherein the human further comprises VEGF-A SNP rs699947 or rs1570360.

In an example, the SNP is rs699947, eg, homozygous for said SNP.

In an example, the SNP is rs1570360, eg, homozygous for said SNP.

Antagonism of VEGF-A & PDGF-B Pathways

PDGF-B is also known as IBGC5, PDGF-2, PDGF2, SIS, SSV and c-sis. Platelet-derived growth factor receptor beta (PDGFRB) is also known as CD140B, IBGC4, IMF1, JTK12, PDGFR-1 and PDGFR1.

PDGF is a growth factor which may play a role in ocular neovascularization, and has been documented to be a mitogen for pericytes, as well as smooth muscles cells, fibroblasts, and other mesenchymal cell types. Pericytes appear critical for the maintenance of established blood vessels and may be important in the maturation process in ocular conditions, such as choroidal neovascularization. It has been hypothesized that there is a period of VEGF dependency of endothelial cells that overlaps with a period of responsiveness to PDGF withdrawal in newly formed neovascular vessels. Established mature vessels may be associated with resistance to anti-VEGF blockade or rapid recurrence of choroidal neovascularization.

PDGF has been demonstrated to stimulate angiogenesis and pericyte recruitment. Loss of pericytes in retinal vessels is thought to be associated with abnormalities of the vasculature and instability, including the formation of microaneurysms and vascular permeability. Loss of pericytes with PDGF-blockade may be associated with regression of maturing neovascularization. Pericytes produce VEGF-A under selected conditions, possibly thereby protecting endothelial cells when generalized suppression of VEGF-A is present.

Reference is made to Am J Pathol. 2006 June; 168(6):2036-53; “Inhibition of platelet-derived growth factor B signaling enhances the efficacy of anti-vascular endothelial growth factor therapy in multiple models of ocular neovascularisation”; Jo N et al. Jo et al demonstrated increased suppression of neovascularization in experimental models with specific blockade of both VEGF-A and PDGF-B as compared with blockade with VEGF-A alone. In addition, neovascularization became more resistant to VEGF-A blockade with progression. Suppression of VEGF-A and PDGF-B together caused regression of neovascularization not responsive to anti-VEGF-A alone.

Reference is also made to:—

-   Angiogenesis. 2014 July; 17(3):553-62. doi:     10.1007/s10456-013-9402-5. Epub 2013 Oct. 24; “Antagonism of PDGF-BB     suppresses subretinal neovascularization and “; enhances the effects     of blocking VEGF-A2; Dong A et al -   MAbs. 2010 January-February; 2(1):20-34. Epub 2010 Jan. 2; “A     dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable     antibody fragments demonstrates anti-angiogenic activity in vitro     and in vivo”; Mabry R et al.

A treatment regimen combining inhibition of pericyte and endothelial cell function through the use of concomitant administration of anti-VEGF-A targeting and PDGF-BB pathway targeting is useful for blocking both paracrine and autocrine VEGF signalling in endothelial cells.

Thus, this aspect of the invention provides the following.

18. The method of any preceding concept, wherein the method further comprises antagonising PDGF-B in said human.

In an example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc) to the human. REGN2176-3 comprises aflibercept.

19. The method of concept 18, wherein the method comprises administering an anti-PDGF-B ligand to the human simultaneously or sequentially with the anti-VEGF-A ligand.

In an example, the anti-VEGF-A and anti-PDGF-B ligands are comprised together by a pharmaceutical formulation that is administered to the human. In an example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc) to the human.

In an example a bispecific ligand (eg, trap or antibody or antibody fragment) is administered, wherein the ligand comprises a human VEGF-A binding site and a human PDGF-B binding site.

In an example, the anti-PDGF-B ligand antagonises a human PDGF-B encoded by said PDGF-B nucleotide sequence of (i).

In an example, the anti-PDGF-B ligand comprises or consists of an anti-PDGF-B antibody or fragment, an anti-PDGF-B trap, an anti-PDGF-B aptamer (eg, E10030 or FOVISTA™) or an anti-PDGF-B NCE (so-called New Chemical Entity in the art).

Examples of suitable anti-PDGF-B pathway antibodies and variable regions for incorporation into antibodies for use in the invention are disclosed in WO2014109999A1, the disclosure of which, and specifically including the sequences therein of such antibodies and variable regions, as well as methods of testing and medical diseases and conditions for use (which are suitable for the present invention), are incorporated herein as though explicitly written herein and to provide disclosure of features that can be incorporated into concepts herein.

In an example herein, the anti-PDGF-B ligand specifically binds to human PDGF-B dimer (ie, so-called PDGF-BB).

20. The method of concept 18, wherein the method comprises administering an anti-PDGFR-B ligand to the human simultaneously or sequentially with the anti-VEGF-A ligand.

In an example, the anti-VEGF-A and anti-PDGFR-B ligands are comprised together by a pharmaceutical formulation that is administered to the human. In an example a bispecific ligand (eg, trap or antibody or antibody fragment) is administered, wherein the ligand comprises a human VEGF-A binding site and a human PDGFR-B binding site. Reference is made to Dong et al, which describes a suitable example.

In an example, the PDGFR-B ligand antagonises a human PDGFR-B encoded by said PDGF-B nucleotide sequence of (ii).

In an example herein, the anti-PDGFRB antagonises binding of the receptor to human PDGF-B dimer (ie, so-called PDGF-BB), eg, wherein the PDGF-B is encoded by a nucleotide sequence of (i).

Suitable PDGFRβ-specific inhibitors are disclosed in WO2008130704, for example, the disclosure and sequences of which are incorporated herein by reference.

21. The method of any preceding concept, wherein the human has received a human PDGF-B or PDGFR-B antagonist prior to administering said anti-VEGF-A ligand.

Antagonism of VEGF-A & Angiopoietin-2 Pathways

The angiopoietins are ligands for the endothelial cell receptor Tie2 and are essential for vascular development and angiogenesis. Unlike other family members, angiopoietin-2 (Ang2) is strongly up-regulated by endothelial cells at sites of angiogenesis and vascular remodelling, including tumours. Enhanced anti-tumour effects have been observed in preclinical models with combined blockade of both VEGF and Ang2.

The Ang2 pathway acts to remodel blood vessels and, in particular, is active during both developmental and pathological angiogenesis. But, it is nearly absent from mature, quiescent vasculature. Hence a combination Ang2/VEGF blockade results in decreased survival and maturation of neovessels as well as inhibition of pathological leaks. Ang2 inhibition also blocks vascular remodelling driven by inflammation in model systems in which VEGF inhibition is ineffective, suggesting that combination blockade has potential for a greater therapeutic benefit.

The invention thus further provides:—

22. The method of any one of concepts 1 to 17, comprising antagonising angiopoietin-2 (Ang2) in said human.

In an example, the method comprises administering nesvacumab (Regeneron Pharmaceuticals, Inc) to the human.

23. The method of concept 22, wherein the method comprises administering an anti-Ang2 ligand (eg, nesvacumab) to the human simultaneously or sequentially with the anti-VEGF-A ligand.

In an example, the anti-Ang2 ligand comprises or consists of an anti-Ang2 antibody or fragment, an anti-Ang2 trap, an anti-Ang2 aptamer or an anti-Ang2 NCE (so-called New Chemical Entity in the art).

24. The method of concept 22, wherein an anti-Ang2 receptor (eg, anti-human Tie2) ligand is administered to the human simultaneously or sequentially with the anti-VEGF-A ligand. 25. The method of any one of concepts 1 to 24, wherein the human has received a human Ang2 or Ang2 receptor (eg, Tie2) antagonist prior to administering said anti-VEGF-A ligand. 26. The method of any preceding concept, wherein the anti-VEGF-A ligand (eg, an anti-VEGF-A antibody, antibody fragment or VEGF-A trap) comprises a human gamma-1 heavy chain constant region that comprises an amino acid selected from the group consisting of an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42; and wherein said human comprises an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising said selected amino acid.

Optionally, this constant region is comprised by an Fc region of the ligand.

In one embodiment, the ligand of the invention is a VEGF-A trap comprising the polypeptide of SEQ ID NO: 127, eg, comprising or consisting of the polypeptide of SEQ ID NO: 127.

27. The method of any one of concepts 1 to 25, wherein the anti-VEGF-A ligand (eg, an anti-VEGF-A antibody, antibody fragment or VEGF-A trap) comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44 and wherein said human comprises an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected amino acid.

Optionally, this constant region is comprised by an Fc region of the ligand.

For example, the ligand is a VEGF-A trap comprising one or more human VEGF-A receptor domain sequences fused to a human gamma-1 or -2 heavy chain constant region, eg, a constant region as defined in concept 26 or 27. In an example, such a trap comprises first and second (eg, comprises only two species of) VEGF-A receptor domain sequences, eg, first and second human Flt1 domain sequences; or first and second human KDR domain sequences; or a Flt domain sequence and a KDR domain sequence. In an example, the trap comprises a dimer of the following polypeptide (N- to C-terminally): a human Flt1 Ig-like C2-type 2 domain sequence and a human KDR Ig-like C2-type 3 domain sequence fused to the human gamma-1 heavy chain constant region of concept 26 or human gamma-2 heavy chain constant region of concept 27. In an example, the constant regions are part of antibody Fc regions.

A suitable human Flt1 Ig-like C2-type 2 domain sequence for use in the present invention is shown in SEQ ID NO: 129. A suitable human KDR Ig-like C2-type 3 domain sequence for use in the present invention is shown in SEQ ID NO: 130.

28. The method of any one of concepts 1 to 27, wherein the anti-VEGF-A ligand is a human anti-VEGF-A antibody, antibody fragment or VEGF-A trap. 29. The method of any preceding concept, comprising, before said administering, selecting a human comprising said VEGF-A, HTRA1, ARMS2, CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP, wherein the human is the human of concept 1 or 12. 30. The method of any one of concepts 1 to 28, wherein the human has been determined to comprise said VEGF-A, HTRA1, ARMS2, CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP. 31. The method of any one of concepts 1 to 28, comprising the step of determining that the human comprises said VEGF-A, HTRA1, ARMS2, CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP, optionally, wherein the determining step is performed before administration of the ligand to the human. 32. The method of concept 31, wherein the step of determining comprises assaying a biological sample from the human for said VEGF-A, HTRA1, ARMS2, CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP. 33. The method of concept 32, wherein the assaying comprises nucleic acid amplification and optionally one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification and/or wherein the assaying is performed in a multiplex format. 34. An anti-VEGF-A ligand (eg, an anti-VEGF-A antibody, antibody fragment or VEGF-A trap) for use in a method of any one of concepts 1 to 33. 35. The ligand of concept 34, wherein the anti-VEGF-A ligand is a human anti-VEGF-A antibody, antibody fragment or VEGF-A trap (eg, aflibercept). 36. The ligand of concept 34 or 35, wherein said ligand is administered by intravenous or subcutaneous administration and/or is comprised in an injectable preparation.

C2 SNP rs547154 is indicated in the 1000 Genomes database as having an average cumulative allele frequency of 10% across all human populations; a frequency for the AFR population of 15% and above average also for the following human populations: PUR, IBS, GBR, ASW, LWK, YRI. Thus, when the ancestry of the human belongs to one of these populations, the human may have an elevated risk of an ocular disease or condition or other VEGF-A mediated disease or condition and the invention comprises administering the ligand to such a human. Thus, in an embodiment, the human comprises C2 SNP rs547154 and is of AFR, PUR, IBS, GBR, ASW, LWK or YRI ancestry. For example, the human is of AFR ancestry.

VEGF-A SNP rs1413711 is indicated in the 1000 Genomes database as having an average cumulative allele frequency of 64% across all human populations; a frequency for the AFR and ASN populations of 80% and 71% respectively, and above average also for the following human populations: ASW, LWK, YRI, PUR, CHB, CHS and JPT. Thus, when the ancestry of the human belongs to one of these populations, the human may have an improved treatment of an ocular disease or condition and the invention comprises administering the ligand to such a human. Thus, in an embodiment, the human comprises VEGF-A SNP rs1413711 (eg, in homozygous form) and is of AFR, ASN, ASW, LWK, YRI, PUR, CHB, CHS or JPT ancestry. For example, the human is of AFR ancestry. For example, the human is of ASN ancestry. In an example, the method is for improving visual acuity in said human.

VEGF-A SNP rs699946 is indicated in the 1000 Genomes database as having an average cumulative allele frequency of 26% across all human populations; a frequency for the ASN population of 42%, and above average also for the following human populations: PUR 34%, CHB 45%, CHS 41% and JPT 40%. Thus, when the ancestry of the human belongs to one of these populations, the human may have an improved treatment of an ocular disease or condition and the invention comprises administering the ligand to such a human. Thus, in an embodiment, the human comprises VEGF-A SNP rs699946 (eg, in homozygous form) and is of ASN, ASW, PUR, CHB, CHS or JPT ancestry. For example, the human is of ASN ancestry. In an example, the method is for improving visual acuity in said human.

VEGF-A SNP rs3025000 is indicated in the 1000 Genomes database as having an average cumulative allele frequency of 26% across all human populations; a frequency for the AMR, ASN and EUR populations of 30%, 42% and 27% respectively, and above average also for the following human populations: MXL (29%), PUR (38%), CHB (45%), CHS (41%, JPT (40%), CEU (30%), GBR (27%), IBS (39%) and TSI (31%). Thus, when the ancestry of the human belongs to one of these populations, the human may have an improved treatment of an ocular disease or condition and the invention comprises administering the ligand to such a human. Thus, in an embodiment, the human comprises VEGF-A SNP rs3025000 (eg, in homozygous form) and is of AMR, ASN, EUR, MXL, PUR, CHB, CHS, JPT, CEU, GBR, IBS or TSI ancestry. For example, the human is of AMR ancestry. For example, the human is of ASN ancestry. For example, the human is of EUR ancestry.

Prothrombin SNP rs1799963 is almost exclusively in Caucasians; this SNP is indicated in the 1000 Genomes database as having an above average cumulative allele frequency in the following human populations: AMR, EUR, TSI, GBR and PUR. Thus, when the ancestry of the human belongs to one of these populations, the human may have an improved treatment of an ocular disease or condition and the invention comprises administering the ligand to such a human. Thus, in an embodiment, the human comprises prothrombin SNP rs1799963 (eg, in homozygous form) and is of Caucasian, AMR, EUR, TSI, GBR or PUR ancestry. For example, the human is of Caucasian ancestry. For example, the human is of EUR ancestry. In an example, the condition is CNV.

MTHFR SNP rs1801133 is indicated in the 1000 Genomes database as having an above average cumulative allele frequency in the following human populations: AMR, ASN, EUR, CHB, CLM, IBS, JPT, MXL, PUR and TSJ. Thus, when the ancestry of the human belongs to one of these populations, the human may have an improved treatment of an ocular disease or condition and the invention comprises administering the ligand to such a human. Thus, in an embodiment, the human comprises MTHFR SNP rs1801133 (eg, in homozygous form) and is of AMR, ASN, EUR, CHB, CLM, IBS, JPT, MXL, PUR or TSI ancestry. For example, the human is of Caucasian ancestry. For example, the human is of AMR ancestry. For example, the human is of ASN ancestry. For example, the human is of EUR ancestry. For example, the human is of EUR ancestry. In an example, the condition is CNV.

VEGFR2 SNP rs4576072 is indicated in the 1000 Genomes database as having an average cumulative allele frequency of 8% across all human populations; a frequency for the AMR and EUR populations of 10% and 11% respectively, and above average also for the following human populations: PUR (14%), CEU (9%), GBR (13%) and TSI (13%). Thus, when the ancestry of the human belongs to one of these populations, the human may have an improved treatment of an ocular disease or condition and the invention comprises administering the ligand to such a human. Thus, in an embodiment, the human comprises VEGFR2 SNP rs4576072 (eg, in homozygous form) and is of AMR, EUR, PUR, CEU, GBR or TSI ancestry. For example, the human is of AMR ancestry. For example, the human is of EUR ancestry. In an embodiment 3 or 4 VEGFR2 SNPs from rs4576072 and rs6828477 provide for improved treatment. Thus, for example, the human comprises 3 or 4 copies of VEGFR2 SNPs selected from rs4576072 and rs6828477, eg, the human comprise GG (ie, the human comprises VEGFR2 SNP rs4576072 in homozygous form) and AA or AG (ie, the human comprises VEGFR2 SNP rs6828477, eg, in homozygous form); or GA (rs4576072) and AA(rs6828477).

Administering Human VEGF-A Ligands to Humans Comprising PDFG-B Pathway SNP Risk Factors

Endothelial cells (ECs) in blood vessels under formation are stabilized by the recruitment of pericytes, both in normal tissues and during angiogenesis in pathologic situations, including cancer. Endothelial cells in small blood vessels and capillaries interact with pericytes, cells of mesenchymal origin that provide important support for blood vessel formation and function. Numerous studies describe the significance of members of the platelet-derived growth factor (PDGF) family for recruitment to and productive association of pericytes with the blood vessel endothelium during embryonal development. Strikingly, mice lacking components of the PDGF-B/PDGFR-$ signaling axis are essentially devoid of pericytes, and as a consequence die from microhemorrhaging because of malformed blood vessels. Reference is made to Blood. 2011 September 8; 118(10): 2906-2917, prepublished online 2011 July 21, doi: 10.1182/blood-2011-01-331694, “Pericytes promote endothelial cell survival through induction of autocrine VEGF-A signaling and Bcl-w expression”, Franco M et al. The authors describe that newly formed vascular sprouts recruit pericytes primarily through secretion of platelet derived growth factor beta (PDGF-BB) by endothelial tip cells. Pericytes respond by migrating along the matrix-retained gradient of PDGF-BB, and by a proliferative burst as they reach the tip of the sprout. Reciprocally, pericytes up-regulate expression of cell-bound VEGF-A as they mature and are recruited into newly formed vessels. The authors found that pericytes, in addition to producing VEGF-A that acts in a paracrine fashion, stimulate autocrine expression of VEGF-A by tumour endothelial cells. The authors suggest that a treatment regimen combining inhibition of pericyte and endothelial cell function through the use of concomitant administration of a VEGF-targeting antibody and a PDGF-BB/PDGFR$-targeting antibody for blocking both paracrine and autocrine VEGF signalling in ECs.

The invention thus provides the following aspects:—

-   1. A method of treating or reducing the risk of a VEGF-A mediated     disease or condition in a human, the method comprising administering     an anti-human VEGF-A ligand; an an anti-human PDGF-B ligand or an     anti-PDGFR-B ligand to a human, wherein the human comprises (i) a     PDGF-B nucleotide sequence comprising a SNP selected from the group     consisting of rs142404523 (ie, a C corresponding to position −776)     and a C at the position of rs1800818 (ie, a C corresponding to     position −735); or (ii) a PDGFR-B nucleotide sequence comprising a     SNP selected from the group consisting of rs246395 (ie, a G     corresponding to position 2601) and rs74943037 (ie, a T     corresponding to position 1391).

A suitable example of an anti-VEGF-A ligand, which is an antagonist of human VEGF-A, is aflibercept, sunitinib, ESBA1008, bevacizumab or ranizumab. In an example, the ligand comprises or consists of an anti-VEGF-A antibody or fragment (eg, ESBA1008, bevacizumab or ranizumab), an anti-VEGF-A trap (eg, VEGF-A receptor binding sites fused to antibody Fc regions, eg, aflibercept), an aptamer or an NCE (so-called New Chemical Entity in the art, eg, sunitinib).

In an example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc) to the human. REGN2176-3 comprises aflibercept and an anti-PDGF-B ligand.

In an embodiment, said selected SNP (eg, rs246395) is associated with VEGF-A secretion from pericytes in said human, for example maintained or increased VEGF-A secretion from pericytes in said human (eg, ocular pericytes or pericytes at the site of a solid tumour in said patient). Thus, administration of an anti-VEGF-A ligand would be beneficial to also target such secreted VEGF-A. This is useful, for example, when the disease or condition is angiogenesis or neovascularisation or associated with one or both of these.

For PDGF-B, a C at the position of rs1800818 (ie, −735C) is indicated in the 1000 Genomes database as having an average cumulative allele frequency of 37% across all human populations; a frequency for the AFR and EUR populations of 62% and 41% respectively (ie, above average) and also above average for the following human populations: ASW 60%, LWK 58%, YRI 68%, CEU 46%, FIN 46%, IBS 50% and TSI 38%. Thus, when the ancestry of the human belongs to one of these populations, the human may have an elevated risk of angiogenesis or other VEGF-A mediated disease or condition and the invention comprises administering the ligand such a human. Thus, in an embodiment, the human comprises a PDGF-B nucleotide sequence comprising −735C, ie, a C at the position of PDGF-B SNP rs1800818 and wherein the human is of AFR, EUR, ASW, LWK, YRI, CEU, FIN, IBS or TSI ancestry. For example, the human is of AFR ancestry. For example, the human is of EUR ancestry.

For PDGF-B, SNP rs142404523 (−776T>C) is indicated in the 1000 Genomes database as having an average cumulative allele frequency of 8% across all human populations; a frequency for the AFR and EUR populations of 17% and 10% respectively (ie, above average) and also above average for the following human populations: ASW 16%, LWK 18%, YRI 16%, PUR 11%, CEU 11%, FIN 9%, GBR 12% and IBS 18%. Thus, when the ancestry of the human belongs to one of these populations, the human may have an elevated risk of angiogenesis or other VEGF-A mediated disease or condition and the invention comprises administering the ligand such a human. Thus, in an embodiment, the human comprises PDGF-B SNP rs142404523 and is of AFR, EUR, ASW, LWK, YRI, PUR, CEU, FIN, GBR or IBS ancestry. For example, the human is of AFR ancestry. For example, the human is of EUR ancestry.

For PDGFR-B, SNP rs246395 (2601A>G) the 1000 Genomes database indicates an average cumulative allele frequency of 23% for G across all human populations; a frequency for the AMR and EUR populations of 29% and 30% respectively (ie, above average) and also above average for the following human populations: CLM (36%), MXL (25%), PUR (26%), CEU (39%), GBR (30%), IBS (29%) and TSI (31%). Thus, when the human belongs to one of these populations, it may have an elevated risk of angiogenesis or other VEGF-A mediated disease or condition and the invention comprises administering the ligand such a human. Thus, in an embodiment, the human comprises PDGFR-B SNP rs246395 and is of AMR, EUR, CLM, MXL, PUR, CEU, GBR, IBS or TSI ancestry. For example, the human is of AMR ancestry. For example, the human is of EUR ancestry.

For PDGFR-B, SNP rs74943037 is indicated in the 1000 Genomes database as having an average cumulative allele frequency of 3% for G across all human populations; a frequency for the AFR population of 10% (ie, above average) and also above average for the following human populations: ASW 8%, LWK 14% and YRI 8%. Thus, when the ancestry of the human belongs to one of these populations, the human may have an elevated risk of angiogenesis or other VEGF-A mediated disease or condition and the invention comprises administering the ligand such a human. Thus, in an embodiment, the human comprises PDGFR-B SNP rs74943037 and is of AFR, ASW, LWK or YRI ancestry. For example, the human is of AFR ancestry.

-   2. The method of aspect 1, wherein the human is homozygous for said     selected SNP (eg, homozygous for rs246395 or homozygous for a C at     the position of rs1800818 (ie, homozygous for a C corresponding to     position −735)).

Such homozygotes may have increased severity or risk of said disease or condition.

-   3. The method of aspect 1 or 2, wherein the human comprises a PDGF-B     nucleotide sequence comprising a SNP of (i) and a PDFGR-B nucleotide     sequence comprising a SNP of (ii).

For example, the human comprises a C at the position of rs1800818 and rs246395. In another example, the human comprises a C at the position of rs1800818 and rs74943037.

For example, the human comprises rs142404523 and rs246395. In another example, the human comprises rs142404523 and rs74943037.

-   4. The method of any one of aspects 1 to 3, wherein the human     comprises (a) a VEGF-A nucleotide sequence comprising a SNP selected     from the group consisting of rs699947, rs833061, rs2010963,     rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069,     rs3025000 and rs1570360; (b) a HTRA1 nucleotide sequence comprising     SNP rs11200638; (c) anARMS2 nucleotide sequence comprising SNP     rs10490924; (d) a Complement Factor H (CFH) nucleotide sequence     comprising SNP rs800292, rs1061170 or rs3766404; (e) a Complement     Component 2 (C2) nucleotide sequence comprising SNP rs547154; (f) a     prothrombin nucleotide sequence comprising SNP rs1799963; (g) a     MTHFR nucleotide sequence comprising SNP rs1801133; (h) a Factor V     nucleotide sequence comprising SNP rs6025; or (i′) a VEGFR2     nucleotide sequence comprising SNP rs4576072 or rs6828477.

In an example, the human comprises a SNP of (a).

For example, the human comprises rs699947 and optionally the condition is DR or a solid tumour.

For example, the human comprises rs833061 and optionally the condition is DR or a solid tumour.

For example, the human comprises rs2010963 and optionally the condition is DR or a solid tumour.

For example, the human comprises rs3025039 and optionally the condition is DR or a solid tumour.

For example, the human comprises rs699946 and optionally the condition is DR or a solid tumour.

For example, the human comprises rs2146323.

For example, the human comprises rs1413711, optionally the human is homozygous for said SNP.

For example, the human comprises rs833068, optionally the human is homozygous for said SNP.

For example, the human comprises rs833069, optionally the human is homozygous for said SNP.

For example, the human comprises rs3025000.

For example, the human comprises rs1570360.

In an example, the human comprises a SNP of (a) and (b). In an example, the human comprises a SNP of (a) and (c). In an example, the human comprises a SNP of (a) and (d). In an example, the human comprises a SNP of (a) and (e). In an example, the human comprises a SNP of (a) and (f). In an example, the human comprises a SNP of (a) and (g). In an example, the human comprises a SNP of (a) and (h). In an example, the human comprises a SNP of (a) and (i′).

Optionally in any aspect herein, the ligand specifically binds to or antagonises a VEGF-A, eg, a VEGF-A encoded by a VEGF-A nucleotide sequence comprising a SNP of (a). In one example, this can be determined by specific binding to or antagonism of VEGF-A in a sample in vitro (eg, serum or blood sample) from the human, wherein the human comprises said VEGF-A nucleotide sequence. Specific binding can be determined, for example, by SPR or ELISA as will be known to the skilled person. The skilled person will also be familiar with standard assays for VEGF-A antagonism. Additionally, in an example the genome of the human comprises said SNP of (a).

-   5. The method of any one of aspects 1 or 4, wherein the human     comprises a human VEGF-A nucleotide sequence comprising a SNP     selected from the group consisting of rs699947, rs833061, rs2010963,     rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069,     rs3025000 and rs1570360 and the ligand antagonises a VEGF-A encoded     by said nucleotide sequence.

In an example of any aspect herein, the VEGF-A is VEGF-A-(165) isoform.

-   6. The method of any one of aspects 1 to 5, wherein the method     further comprises antagonising PDGF-B in said human.

In an example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc) to the human. REGN2176-3 comprises aflibercept.

-   7. The method of aspect 6, wherein the method comprises     administering an anti-PDGF-B ligand to the human simultaneously or     sequentially with the anti-VEGF-A ligand.

In an example, the anti-VEGF-A and anti-PDGF-B ligands are comprised together by a pharmaceutical formulation that is administered to the human. In an example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc) to the human.

In an example, the anti-PDGF-B ligand antagonises a human PDGF-B encoded by said PDGF-B nucleotide sequence of (i).

In an example, the anti-PDGF-B ligand comprises or consists of an anti-PDGF-B antibody or fragment, an anti-PDGF-B trap, an anti-PDGF-B aptamer (eg, E10030 (FOVISTA™), described in, e.g., U.S. Pat. Nos. 6,207,816; 5,731,144; 5,731,424; and 6,124,449; each of which is incorporated by reference herein in its entirety) or an anti-PDGF-B NCE (so-called New Chemical Entity in the art).

Examples of suitable anti-PDGF-B pathway antibodies and variable regions for incorporation into antibodies for use in the invention are disclosed in WO2014109999A1, the disclosure of which, and specifically including the sequences therein of such antibodies and variable regions, as well as methods of testing and medical diseases and conditions for use (which are suitable for the present invention), are incorporated herein as though explicitly written herein and to provide disclosure of features that can be incorporated into aspects herein.

In an example herein, the anti-PDGF-B ligand specifically binds to human PDGF-B dimer (ie, so-called PDGF-BB).

-   8. The method of aspect 6, wherein the method comprises     administering an anti-PDGFR-B ligand to the human simultaneously or     sequentially with the anti-VEGF-A ligand.

In an example, the anti-VEGF-A and anti-PDGFR-B ligands are comprised together by a pharmaceutical formulation that is administered to the human.

In an example, the PDGFR-B ligand antagonises a human PDGFR-B encoded by said PDGF-B nucleotide sequence of (ii).

In an example herein, the anti-PDGFRB antagonises binding of the receptor to human PDGF-B dimer (ie, so-called PDGF-BB), eg, wherein the PDGF-B is encoded by a nucleotide sequence of (i).

Suitable PDGFRβ-specific inhibitors are disclosed in WO2008130704, for example, the disclosure and sequences of which are incorporated herein by reference.

-   9. The method of any one of aspects 1 to 8, wherein the human has     received a human PDGF-B or PDGFR-B antagonist prior to administering     said anti-VEGF-A ligand. -   10. The method of any one of aspects 1 to 9, wherein the condition     is selected from the group consisting of angiogenesis,     neovascularisation, ocular vascularisation and solid tumour     vascularisation.

In an example, the condition is subretinal neovascularisation.

-   11. The method of any one of aspects 1 to 9, wherein the disease or     condition is an angiogenic disease or condition.

Optionally the disease or condition is an angiogenic ocular condition or angiogenic cancer, eg a solid tumour, a gastrointestinal cancer, colorectal cancer, liver cancer, breast cancer, ovarian cancer, non small cell lung cancer, thyroid cancer, an oral cancer, or an haematological cancer.

Optionally the angiogenic disease or condition is selected from the group consisting of psoriasis, rheumatoid arthritis, a hemangioma, an angiofibroma, diabetic retinopathy, corneal neovacularisation and neovascular glaucoma.

-   12. The method of any one of aspects 1 to 11, wherein the disease or     condition is or comprises vascular permeability (eg, ocular vascular     permeability), edema or inflammation in said human.

For example, the disease or condition is selected from the group consisting of brain edema (eg, edema associated with brain injury, stroke or brain tumour); edema associated with an inflammatory disorder (eg, associated with psoriasis or arthritis, rheumatoid arthritis or asthma); edema associated with burns; ascites or pleural effusion (eg, associated with tumours, inflammation or trauma); chronic airway inflammation; capillary leak syndrome; sepsis; kidney disease (eg, associated with increased leakage of protein); and an eye disorder (eg, age related macular degeneration or diabetic retinopathy).

-   13. The method of any one of aspects 1 to 12 for reducing or     reducing the risk of VEGF-A mediated endothelial cell maintenance or     proliferation in an angiogenic disease or condition in said human.

In an example, the angiogenic disease or condition is an angiogenic disease or condition recited above, eg, an angiogenic ocular condition.

-   14. The method of any one of aspects 1 to 12 for regressing     neovascularisation in a VEGF-A mediated disease or condition in said     human. -   15. The method of any one of aspects 1 to 12 for treating or     reducing the risk of neovascularisation in a human, wherein the     human has VEGF-A ligand-refractory neovascularisation.

For example, the human is partially or completely resistant to treatment with an anti-VEGF-A ligand, eg, aflibercept, sunitinib, bevacizumab or ranizumab.

-   16. The method of any one of aspects 1 to 15, comprising     antagonising angiopoietin-2 (Ang2) in said human.

The angiopoietins are ligands for the endothelial cell receptor Tie2 and are essential for vascular development and angiogenesis. Unlike other family members, angiopoietin-2 (Ang2) is strongly up-regulated by endothelial cells at sites of angiogenesis and vascular remodelling, including tumours. Enhanced anti-tumour effects have been observed in preclinical models with combined blockade of both VEGF and Ang2.

In an example, the method comprises administering nesvacumab (Regeneron Pharmaceuticals, Inc) to the human.

-   17. The method of aspect 16, wherein the method comprises     administering an anti-Ang2 ligand (eg, nesvacumab) to the human     simultaneously or sequentially with the anti-VEGF-A ligand.

In an example, the anti-Ang2 ligand comprises or consists of an anti-Ang2 antibody or fragment, an anti-Ang2 trap, an anti-Ang2 aptamer or an anti-Ang2 NCE (so-called New Chemical Entity in the art).

-   18. The method of aspect 16, wherein an anti-Ang2 receptor (eg,     anti-human Tie2) ligand is administered to the human simultaneously     or sequentially with the anti-VEGF-A ligand. -   19. The method of any one of aspects 1 to 18, wherein the human has     received a human Ang2 or Ang2 receptor (eg, Tie2) antagonist prior     to administering said anti-VEGF-A ligand. -   20. An anti-TOI (eg, VEGF-A) ligand (eg, an antibody, antibody     fragment or TOI trap) for use in a method of any one of aspects 1 to     19. -   21. The ligand of aspect 20, wherein the ligand is a human     anti-VEGF-A antibody, antibody fragment or VEGF-A trap (eg,     aflibercept). -   22. The ligand of aspect 20 or 21, wherein said ligand is for     intravenous or subcutaneous administration and/or is comprised in an     injectable preparation.

General Optional Features Applicable to All Examples, Embodiments, Concepts & Aspects

There is optionally provided the method or ligand wherein the anti-TOI (eg, VEGF-A) ligand (eg, an antibody, antibody fragment or TOI trap) comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising an Asp corresponding to position 204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42.

For example, the ligand is a VEGF-A trap comprising one or more human VEGF-A receptor domain sequences fused to a human gamma-1 heavy chain constant region, eg, a constant region as defined in the immediately preceding paragraph. In an example, such a trap comprises first and second (eg, comprises only two) VEGF-A receptor domain sequences, eg, first and second human Flt1 domain sequences; or first and second human KDR domain sequences; or a Flt domain sequence and a KDR domain sequence. In an example, the trap comprises a human Flt1 Ig-like C2-type 2 domain sequence and a human KDR Ig-like C2-type 3 domain sequence (eg, in this order in the N- to C-terminal direction).

In one embodiment, the ligand of the invention is a VEGF-A trap comprising or of SEQ ID NO: 127, eg, comprising or consisting of a dimer of SEQ ID NO: 127.

In an example, the condition is an ophthalmic angiogenic disorder.

In an example, the condition is selected from the group consisting of AMD, age-related macular degeneration; BRVO, branch retinal vein occlusion; CNV, choroidal neovascularization; mCNV, choroidal neovascularization caused by pathologic myopia; CRVO, central retinal vein occlusion; DME, diabetic macular edema; glaucoma; Macular edema; PCV, polypoidal choroidal vasculopathy; RAP, retinal angiomatous proliferation; retinal neovascularisation; ROP, retinopathy of prematurity; and RVO, retinal vein occlusion.

In an example, the condition is exudative AMD.

In an example, the method is for the treatment of Adult-Onset Vitelliform Detachments Associated With Pattern Dystrophy.

In an example, the method is for the treatment of Pattern Dystrophy.

In an example, the method is for the treatment of Sub-Retinal Fibrosis in Neovascular AMD, eg, by administering Fovista® (an anti-PDGF BB) plus the anti-VEGF ligand (eg, aflibercept, becavizumab or ranibizumab).

In an example, the human is aged >50 years, eg, the human is more than 55, 60, 65 or 70 years of age.

In an example, the condition is an ocular condition and the anti-VEGF-A ligand is administered in combination with photodynamic therapy (PDT), eg, for treating or preventing PCV.

In an example, the condition is an ocular condition and the anti-VEGF-A ligand is administered in combination with laser treatment and/or IVTA treatment, eg, for treating or preventing DME.

In an example, the condition is an ocular condition and the anti-VEGF-A ligand is administered in combination with laser photocoagulation and/or intravitreal injections of a glucocorticoid.

In an example, the condition is an ocular condition and the anti-VEGF-A ligand is administered in combination with PDT and triamcinolone acetonide. Optionally, the ligand is administered intravitreally.

In an example the human is suffering from hypertension, hyperlipidemia, diabetes mellitus and/or arteriosclerotic vascular disorder. Optionally, the condition is RVO and further optionally the human is greater than 60 years' of age.

In an example, when RVO is mentioned herein, the RVO is ischemic (non-perfusion) or non-ischemic (perfusion) RVO.

In an example, when ROP is mentioned herein, the type-1 ROP.

In an example, the condition is ROP and the anti-VEGF-A ligand is administered in combination with laser treatment.

In an embodiment, the human has a Body Mass Index (BMI) >30 and/or the human is a cigarette smoker. These are risk factors, eg, wherein the disease or condition is an ocular disease or condition.

Optionally there is less than 3 or 2 or 2 weeks between ocular disease or condition symptom onset and initiation of treatment with the ligand according to the invention.

In an example, the AMD is neovascular (nv) AMD, “wet” AMD or atrophic or “dry” AMD.

In an example, the ocular condition or disease (eg, AMD) is stage IV disease according to the Age-Related Eye Disease Study classification (see, eg, Augood C A et al, “Prevalence of agerelated maculopathy in older Europeans: the European Eye Study (EUREYE)”, Arch Ophthalmol. 2006; 124:529e35) when the ligand is administered to the human.

In an embodiment, wherein the disease or condition is an ocular disease or condition (eg, AMD, PCV, DR or CNV), the ligand is administered to the human by intraocular injection, eg, intravitreal injection.

Persistent angiogenesis may cause or exacerbate certain diseases such as psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy and neovascular glaucoma. An inhibitor of VEGF activity would be useful as a treatment for such diseases and other VEGF-induced pathological angiogenesis and vascular permeability conditions, such as tumor vascularization. Thus, in an embodiment the present invention in Example 5 provides a method (or ligand for use in the method) for treating or reducing the risk of a condition associated with angiogenesis, wherein the condition is psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy or neovascular glaucoma. In an embodiment the present invention in Example 5 provides a method (or ligand for use in the method) for treating or reducing the risk of tumor vascularization.

By way of example, but not limitation, the method of the invention may be useful in treating a clinical conditions that are characterized by vascular permeability, edema or inflammation such as brain edema associated with injury, stroke or tumor; edema associated with inflammatory disorders such as psoriasis or arthritis, including rheumatoid arthritis; asthma; generalized edema associated with burns; ascites and pleural effusion associated with tumors, inflammation or trauma; chronic airway inflammation; capillary leak syndrome; sepsis; kidney disease associated with increased leakage of protein; and eye disorders such as age related macular degeneration and diabetic retinopathy. Thus, in an embodiment the present invention in Example 5 provides a method (or ligand for use in the method) for treating or reducing the risk of any one of these conditions.

In an example, the method of the present invention comprises sequentially administering multiple doses of a VEGF antagonist to a patient. The methods of the present invention include the administration of multiple doses of a VEGF antagonist to a patient at a frequency of once every 8 or more weeks.

Various administration routes are contemplated for use in the methods of the present invention, including, e.g., topical administration or intraocular administration (e.g., intravitreal administration). The ligand (or pharmaceutical formulation comprising the ligand) may be administered to the patient by any known delivery system and/or administration method. In certain embodiments, the ligand is administered to the patient by ocular, intraocular, intravitreal or subconjunctival injection. In other embodiments, the ligand can be administered to the patient by topical administration, e.g., via eye drops or other liquid, gel, ointment or fluid which contains the ligand and can be applied directly to the eye. Other possible routes of administration include, e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral.

The amount of anti-VEGF ligand administered to the patient in each dose is, in most cases, a therapeutically effective amount. As used herein, the phrase “therapeutically effective amount” means a dose of ligand that results in a detectable improvement in one or more symptoms or indicia of an angiogenic eye disorder, or a dose of ligand that inhibits, prevents, lessens, or delays the progression of an angiogenic eye disorder. In the case of an anti-VEGF antibody or a VEGF receptor-based chimeric molecule (eg, VEGF trap, eg, aflibercept) such as VEGFR1R2-FcAC1(a), a therapeutically effective amount can be from about 0.05 mg to about 5 mg, e.g., about 0.05 mg, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 1.0 mg, about 1.05 mg, about 1.1 mg, about 1.15 mg, about 1.2 mg, about 1.25 mg, about 1.3 mg, about 1.35 mg, about 1.4 mg, about 1.45 mg, about 1.5 mg, about 1.55 mg, about 1.6 mg, about 1.65 mg, about 1.7 mg, about 1.75 mg, about 1.8 mg, about 1.85 mg, about 1.9 mg, about 2.0 mg, about 2.05 mg, about 2.1 mg, about 2.15 mg, about 2.2 mg, about 2.25 mg, about 2.3 mg, about 2.35 mg, about 2.4 mg, about 2.45 mg, about 2.5 mg, about 2.55 mg, about 2.6 mg, about 2.65 mg, about 2.7 mg, about 2.75 mg, about 2.8 mg, about 2.85 mg, about 2.9 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, or about 5.0 mg of the antibody or receptor-based chimeric molecule.

Dosing Regimens

Specific, non-limiting examples of dosing regimens within the scope of the present invention are as follows for an anti-TOI ligand (eg, anti-VEGF-A antibody or trap, eg, aflibercept):

-   -   a. ligand 2 mg (0.05 mL) administered by intravitreal injection         once every 4 weeks (monthly).     -   b. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05         mL) via intravitreal injection once every 8 weeks.     -   c. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05         mL) via intravitreal injection on a less frequent basis based on         visual and/or anatomical outcomes (as assessed by a physician or         other qualified medical professional).     -   d. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05         mL) via intravitreal injection administered pro re nata (PRN)         based on visual and/or anatomical outcomes (as assessed by a         physician or other qualified medical professional).     -   e. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 12 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection once every 8 weeks.     -   f. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 12 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection on a less frequent basis         based on visual and/or anatomical outcomes (as assessed by a         physician or other qualified medical professional).     -   g. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 12 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection administered pro re nata         (PRN) based on visual and/or anatomical outcomes (as assessed by         a physician or other qualified medical professional).     -   h. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 16 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection once every 8 weeks.     -   i. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 16 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection on a less frequent basis         based on visual and/or anatomical outcomes (as assessed by a         physician or other qualified medical professional).     -   j. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 16 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection administered pro re nata         (PRN) based on visual and/or anatomical outcomes (as assessed by         a physician or other qualified medical professional).     -   k. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 20 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection once every 8 weeks.     -   l. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 20 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection on a less frequent basis         based on visual and/or anatomical outcomes (as assessed by a         physician or other qualified medical professional).     -   m. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 20 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection administered pro re nata         (PRN) based on visual and/or anatomical outcomes (as assessed by         a physician or other qualified medical professional).     -   n. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 24 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection once every 8 weeks.     -   o. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 24 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection on a less frequent basis         based on visual and/or anatomical outcomes (as assessed by a         physician or other qualified medical professional).     -   p. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 24 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection administered pro re nata         (PRN) based on visual and/or anatomical outcomes (as assessed by         a physician or other qualified medical professional).     -   q. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 28 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection once every 8 weeks.     -   r. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 28 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection on a less frequent basis         based on visual and/or anatomical outcomes (as assessed by a         physician or other qualified medical professional).     -   s. ligand 2 mg (0.5 mL) administered by intravitreal injection         once every 4 weeks for the first 28 weeks, followed by 2 mg         (0.05 mL) via intravitreal injection administered pro re nata         (PRN) based on visual and/or anatomical outcomes (as assessed by         a physician or other qualified medical professional).     -   t. ligand 2 mg (0.05 mL) administered by intravitreal injection         as a single initial dose, followed by additional doses         administered pro re nata (PRN) based on visual and/or anatomical         outcomes (as assessed by a physician or other qualified medical         professional).

Variations on the above-described dosing regimens would be appreciated by persons of ordinary skill in the art and are also within the scope of the present invention. For example, the amount of ligand and/or volume of formulation administered to a patient may be varied based on patient characteristics, severity of disease, and other diagnostic assessments by a physician or other qualified medical professional.

When the method or ligand is for treating or reducing the risk of an ocular condition or disease, the method in an example improves the Best Correct Visual Acuity in the human.

Best Corrected Visual Acuity:

Visual function of the eye can be assessed using the ETDRS protocol (The Early Treatment Diabetic Retinopathy Study Group) at 4 meters. Visual Acuity examiners are typically certified to ensure consistent measurement of BCVA.

The method in an example method prevents vision loss of greater than or equal to 15 letters on the ETDRS chart, compared to baseline, at 52 weeks. Additionally or alternatively the method provides one or more of the following: (a) change from baseline to Week 52 in letter score on the ETDRS chart; (b) gain from baseline to Week 52 of 15 letters or more on the ETDRS chart; (c) change from baseline to Week 52 in total NEI VFQ-25 score; and (d) change from baseline to Week 52 in CNV area (eg, where the condition is CNV).

In one embodiment, the ligand is an VEGF antagonist that comprises one or more VEGF receptor-based chimeric molecule(s), (also referred to herein as a “VEGF-Trap” or “VEGFT”). An exemplary VEGF antagonist that can be used in the context of the present invention is a multimeric VEGF-binding protein comprising two or more VEGF receptor-based chimeric molecules referred to herein as “VEGFR1R2-FcAC1(a)” or “aflibercept.”

Various ligand or antibody administration routes are contemplated for use in the methods of the present invention, including, e.g., topical administration or intraocular administration (e.g., intravitreal administration).

The methods of the invention optionally comprise sequentially administering to a patient multiple doses of a ligand, eg, VEGF antagonist. As used herein, “sequentially administering” means that each dose of ligand is administered to the patient at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of a ligand, followed by one or more secondary doses of the ligand, followed by one or more tertiary doses of the ligand.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the ligand. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of ligand, but will generally differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of ligand contained in the initial, secondary and/or tertiary doses will vary from one another (e.g., adjusted up or down as appropriate) during the course of treatment.

Example 6: PD-L1 & PD-1

Application of PD-L1 Variation in the Present Invention

Human PD-L1 is also known as CD274, B7-H, B7H1, B7-H1, B7 homolog 1, MGC142294, MGC142296, PDCDIL1, PDCDILG1, PDCD1 ligand 1, PDL1, Programmed cell death 1 ligand 1 and Programmed death ligand 1; and has Uniprot number Q9NZQ7 and NCBI gene ID number 29126. PD-L1 is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on human chromosome 9. PD-L1 expression is implicated in evasion of immune responses involved in chronic infection, e.g., by viruses (including, for example, HIV, HBV, HCV and HTLV, among others), by bacteria (including, for example, Helicobacter pylori, among others) and by parasites (including, for example, Schistosoma mansoni). CD274/PD-L1 expression is also implicated in suppression of anti-tumour immune activity. Tumours express antigens that can be recognized by host T cells, but immunologic clearance of tumours is rare. Part of this failure is due to immune suppression by the tumour microenvironment. PD-L1 expression on many tumours is a component of this suppressive milieu and may act in concert with other immunosuppressive signals. PD-L1 expression has been shown in situ on a wide variety of solid tumours including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck (Brown J A et al., 2003. J. Immunol. 170:1257-66; Dong H et al. 2002. Nat. Med. 8:793-800; Hamanishi J, et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65; Strome S E et al. 2003. Cancer Res. 63:6501-5; Inman B A et al. 2007. Cancer 109:1499-505; Konishi J et al. 2004. Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007. Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007. Clin. Cancer Res. 13:2151-57; Thompson R H et al. 2004. Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C, Zhu Y, Jiang J, Zhao J, Zhang X G, Xu N. 2006. Acta Histochem. 108:19-24). In addition, PD-1 expression is upregulated on tumour infiltrating lymphocytes, and this may also contribute to tumour immunosuppression (Blank C et al. 2003. J. Immunol. 171:4574-81). In ovarian cancer, PD-L1 expression is inversely correlated with intraepithelial, but not stromal, infiltrating CD8 T cells, suggesting that PD-L1 inhibits the intratumor migration of CD8 T cells (Hamanishi J et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65). Translation of PD-L1 mRNA is enhanced by loss of PTEN and the ensuing activation of Akt, a common event in tumourigenesis (Parsa A T et al. 2007. Nat. Med. 13:84-88). Most importantly, studies relating PD-L1 expression on tumours to disease outcome show that PD-L1 expression strongly correlates with unfavorable prognosis in kidney, ovarian, bladder, breast, gastric, and pancreatic cancer (Hamanishi J et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65; Inman B A et al. 2007. Cancer 109:1499-505; Konishi J et al. 2004. Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007. Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007. Clin. Cancer Res. 13:2151-57; Thompson R H et al. 2004. Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C, Zhu Y, Jiang J, Zhao J, Zhang X G, Xu N. 2006. Acta Histochem. 108:19-24). In addition, these studies suggest that higher levels of PD-L1 expression on tumours may facilitate advancement of tumour stage and invasion into deeper tissue structures.

The PD-1 pathway can also play a role in hematologic malignancies. PD-L1 is expressed on multiple myeloma cells but not on normal plasma cells (Liu J et al. 2007. Blood 110:296-304). PD-L1 is expressed on some primary T cell lymphomas, particularly anaplastic large cell T lymphomas (Brown J A et al., 2003. J. Immunol. 170:1257-66). PD-1 is highly expressed on the T cells of angioimmunoblastic lymphomas, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman D M et al. 2006. Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, the T cells associated with lymphocytic and/or histiocytic (L&H) cells express PD-1. Microarray analysis using a readout of genes induced by PD-1 ligation suggests that tumour-associated T cells are responding to PD-1 signals in situ in Hodgkin lymphoma (Chemnitz J M et al. 2007. Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T cell leukemia and lymphoma (Shimauchi T et al. 2007. Int. J. Cancer 121: 2585-90). These tumour cells are hyporesponsive to TCR signals.

Studies in animal models demonstrate that PD-L1 on tumours inhibits T cell activation and lysis of tumour cells and in some cases leads to increased tumour-specific T cell death (Dong H et al. 2002. Nat. Med. 8:793-800; Hirano F et al. 2005. Cancer Res. 65:1089-96). Tumour-associated APCs can also utilize the PD-1:PD-L pathway to control antitumor T cell responses. PD-L1 expression on a population of tumour-associated myeloid DCs is upregulated by tumour environmental factors (Curiel T J et al. 2003. Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in the tumour-draining lymph node of B16 melanoma express IDO, which strongly activates the suppressive activity of regulatory T cells. The suppressive activity of IDO-treated regulatory T cells required cell contact with IDO-expressing DCs (Sharma M D et al. 2007. J. Clin. Invest. 117:2570-82).

U.S. Pat. No. 8,507,663 discusses the specific inhibition of the expression of the CD274/PD-L1 gene. Reference is also made to WO2011066389, WO2010036959, WO 2010077634, WO 03099196, WO 2013181634, WO2007005874, US20090317368 and U.S. Pat. No. 7,635,757.

The inventor carried out natural human variation in PD-L1 and identified the following variations of interest for application to the present invention.

TABLE 21 Human PD-L1 Variation CHROMOSOMAL LOCATION AMINO AMINO (FORWARD NUCLEOTIDE ACID ACID CUMULATIVE SNP/VARIANT STRAND) CHANGE¹ CHANGE² POSITION³ FREQUENCY⁴ rs150439231 9: 5448539 rs138261640 9: 5448573 rs142983488 9: 5448634 rs76741468 9: 5448641 rs822336 9: 5448690 rs183400620 9: 5448763 rs187252832 9: 5448815 rs146143976 9: 5448859 rs139023765 9: 5448896 rs73641615 9: 5448956 rs17718883 9: 5462876 P/R 146 rs139709512 9: 5456126 A/P 5 rs140045210 9: 5462881 T/S 148 rs141978642 9: 5466785 V/E 269 rs146495642 9: 5462929 G > A E/K 164 rs370800260 9: 5457087 G > A V/I 21 rs143235887 9: 5457091 C > T T/M 22 rs373692552 9: 5457117 G > C E/Q 31 rs140304675 9: 5457127 G > C S/T 34 rs12551333 9: 5457171 G > C D/H 49 rs376993991 9: 5457282 C > T R/W 86 rs367921713 9: 5462893 G > A E/K 152 rs41280721 9: 5462997 A > C R/S 186 rs61752860 9: 5465514 A > T H/L 233 rs148141792 9: 5465595 G > A R/H 260 rs369350813 9: 5467858 C > T T/M 290 rs10481593 9: 5458095 rs2282055 9: 5455732 rs2297135 9: 5470108 rs2297136 9: 5467955 rs2297137 9: 5465732 rs3780395 9: 5464552 rs7023227 9: 5456550 rs1411262 9: 5459419 8923 A > C C > T rs4143815 9: 5468257 6777 C > G 395 G > C rs34028061 9: 5454629-5454630 AT>- deletion 0.476 rs7041009 9: 5463243 G > A 0.459 rs148170925 9: 5454642-5454643 TG>- deletion 0.600 rs1411262 9: 5459419 C/T 0.395 rs10114060 9: 5461729 G/A 0.382 rs1536926 9: 5463988 G/T 0.360 rs7042084 9: 5458035 G/T 0.352 ^(1,2,3,4)According to Ensembl, dbSNP; with reference to transcript ENST00000381577 (the sequence of this transcript being incorporated herein in its entirety).

The table is to read as follows: For example, the following for rs12551333 means Asp49His, ie, a histidine at position 49 encoded by SNP rs12551333 D/H 49

REFERENCES

-   1. Asia Pac J Clin Oncol. 2014 June; 10(2):el-6. doi:     10.1111/ajco.12037. Epub 2012 Nov. 21; “Association between single     nucleotide polymorphism of PD-L1 gene and non-small cell lung cancer     susceptibility in a Chinese population”; Chen Y et al.

The authors published the following:—

Aim:

To evaluate the correlation between a polymorphism of PD-L1 gene and the susceptibility of non-small cell lung cancer (NSCLC) in a Chinese population.

Methods:

A total of 293 Chinese patients with NSCLC and 293 age and sex matched controls of the same ethnic origin were enrolled in this study. A/C polymorphism at position 8923 in intron 4 of PD-L1 gene was typed using the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). The interactions between A/C genotype, allele frequency and NSCLC susceptibility were analyzed.

Results:

The A/C genotype frequencies were significantly different between NSCLC patients and controls. The AC and CC frequencies were higher in NSCLC patients than in controls (16.4 vs 8.9%, 1.0 vs 0.3%, respectively). The C-allele frequency was higher in NSCLC patients than in controls (9.2 vs 4.8%). Significant differences in the A and C allele frequencies were noted between the two groups (χ(2)=8.864, P=0.003). More risk of NSCLC was found in individuals carrying the C allele than in those carrying the A allele (OR=2.203; 95% CI 1.262-3.242). In both light smokers (<20 pack-years) and heavy smokers (>20 pack-years), individuals carrying the C-allele had more risk of NSCLC than those carrying the A-allele (light smokers OR=1.847, 95% CI 1.001-3.409; heavy smokers OR=3.252, 95% CI 1.196-8.845, respectively).

Conclusion:

An A/C polymorphism at position 8923 in the PD-L1 gene is associated with NSCLC susceptibility. The PD-L1 polymorphism plays a role in NSCLC, especially in patients with the C-allele.

-   2. Eur J Endocrinol. 2008 June; 158(6):817-22. doi:     10.1530/EJE-07-0649. Epub 2008 Mar. 5. “Association of an A/C single     nucleotide polymorphism in programmed cell death-ligand 1 gene with     Graves' disease in Japanese patients”; Hayashi M et al.

The authors published the following:—

OBJECTIVE: Programmed cell death-1 (PD-1) and its ligands (PD-L1 and PD-L2) inhibit T-cell proliferation and activation. This inhibition down-regulates the immune responses. The association of a PD-L1 polymorphism with Graves' disease (GD) was studied.

DESIGN: The association of an A/C polymorphism at position 8923 in PD-L1 intron 4 with GD was studied.

PATIENTS: The study included 327 GD patients and 192 controls, of which 252 GD patients were followed over 5-10 years.

MEASUREMENTS: PD-L1 intron 4 position 8923 A/C polymorphism was typed using the PCR-restriction fragment length polymorphism method.

RESULTS: The A/C genotype frequencies were significantly different between GD patients and controls. The A/C and C/C frequencies were higher in GD patients than in controls. The A/A frequencies were lower in GD patients than in controls. C-allele frequency was higher in GD patients than in controls. A total of 252 GD patients were followed over 5-10 years; 200 had discontinued antithyroid drugs (ATD) while 52 continued to take ATD. Of these 200, 176 continued to be in remission and 24 had relapsed into hyperthyroidism. Significant differences in the duration of positive TBII, positive thyroid-stimulating antibodies, and ATD treatment were noted between the patients in remission and those that had relapsed. Significant differences in the A- and C-allele frequencies were noted between the two. The C-allele frequency was higher in GD patients who did not achieve remission than in those who achieved remission.

CONCLUSION: An A/C polymorphism at position 8923 in PD-L1 is associated with GD. The PD-L1 polymorphism plays a role in GD development. GD patients with the C allele at position 8923 in PD-L1 gene had difficulty in achieving remission.

-   3. Rheumatology (Oxford). 2011 October; 50(10):1809-13. doi:     10.1093/rheumatology/ker211. Epub 2011 Jul. 26; “Effects of genetic     polymorphisms of programmed cell death 1 and its ligands on the     development of ankylosing spondylitis”; Huang C et al.

Human subjects with the PD-1 GG genotype had significantly greater risk for ankylosing spondylitis (AS) than those with the AA genotype. Subjects with the PD-L2 CT genotype had lower risk for AS than those with the CC genotype. The combined genotypes of PD-1 G-536A, PD-L1 A8923C and PD-L2 C47103T also appear to be associated with AS development.

CONCLUSIONS. The results suggest that PD-1 G-536A, PD-L1 A8923C and PD-L2 C47103T polymorphisms are associated with the presence of AS.

-   4. J Clin Endocrinol Metab. 2009 December; 94(12):5139-45. doi:     10.1210/jc.2009-1404. Epub 2009 Oct. 22; “Programmed death ligand 1     (PD-L1) gene variants contribute to autoimmune Addison's disease and     Graves' disease susceptibility”; Mitchell A L et al.

The authors published the following:—

Context:

Despite much investigation, a substantial amount of the genetic susceptibility to autoimmune diseases remains unaccounted for. Recently, a single-nucleotide polymorphism (SNP) in the programmed death ligand 1 (PD-L1) gene has been associated with Graves' disease (GD) in a Japanese patient cohort. Our aim was to determine whether variants in PD-L1 are also associated with autoimmune Addison's disease (AAD) and to replicate the previous association in patients with GD from the United Kingdom.

Design and Patients:

We analyzed eight SNPs within PD-L1 in a United Kingdom cohort of 315 AAD subjects and 316 healthy controls. We then replicated our experiment in a cohort of 342 Norwegian AAD cases and 379 controls and in 496 United Kingdom GD subjects.

Results:

Three of the eight SNPs studied, part of a haplotype block in the PD-L1 gene, showed modest association with both AAD and GD in the United Kingdom cohort, with maximum evidence at the marker RS1411262 [United Kingdom AAD odds ratio 1.33 (5-95% confidence interval 1.02-1.73), P(genotype)=0.028; GD odds ratio 1.36 (5-95% confidence interval 1.07-1.72), P(genotype)=0.033]. Association with genotypes at the same three markers was confirmed in the Norwegian AAD cohort [P(genotype)=0.011-0.020]. A recessive effect at the most associated alleles was observed in both the AAD and GD cohorts.

Conclusions:

We confirm the role of PD-L1 variants in GD susceptibility and extend these findings to demonstrate association in two Northern European patient cohorts with AAD. PD-L1 joins the growing number of known susceptibility loci exerting modest effects in these autoimmune disorders.

-   5. J Clin Immunol. 2007 November; 27(6):563-7. Epub 2007 Jun. 28;     “Polymorphisms of genes for programmed cell death 1 ligands in     patients with rheumatoid arthritis”; Wang S C et al.

The authors published the following:—

To investigate the role of ligands for programmed cell death 1 (PD-L) in the pathogenesis of rheumatoid arthritis (RA), 129 patients with RA and 125 unrelated healthy controls were enrolled in this study. The PD-L1 and PD-L2 polymorphisms were determined by the method of polymerase chain reaction (PCR)/direct sequencing or PCR/reaction fragment length polymorphisms. The genotype distributions of PD-L1 6777 C/G were not significantly different between the patients with RA and healthy controls. There was also no significant difference in the allele frequencies of PD-L1 6777 C/G polymorphisms between the patients with RA and controls. Similar findings could also be found in the phenotypes and alleles frequencies of PD-L2 47103 C/T and 47139 T/C polymorphisms between the patients with RA and controls. The patients with PD-L1 6777 G had higher prevalence of rheumatoid nodule in comparison with those without PD-L1 6777 G (p=0.005, OR=4.0, 95% CI=1.5-10.9). In contrast, the PD-L2 47103 C/T and 47139 T/C polymorphisms were not related to the occurrence of rheumatoid nodule. This study demonstrated that the PD-L1 and PD-L2 polymorphisms were not associated with susceptibility to RA in Taiwan. PD-L1 6777 G was associated with the prevalence of rheumatoid nodule.

-   6. Hum Genet. 2013 June; 132(6):641-8. doi:     10.1007/s00439-013-1275-6. Epub 2013 Feb. 21; “A miR-570 binding     site polymorphism in the B7-H1 gene is associated with the risk of     gastric adenocarcinoma”; Wang W et al.

Since the over-expression of B7-H1 protein has been reported to be closely related to disease progression of gastric cancer, the authors investigated the possible role of miRSNPs at the 3′-untranslated region (3′-UTR) of B7-H1 in the risk of developing gastric cancer. In this association study on 205 gastric adenocarcinoma patients and 393 non-cancer controls, they found that the genotype distribution of a common C>G polymorphism (rs4143815) was significantly different between the cases and controls (P=1.32×10(−8)). Compared with CC homozygotes, GG homozygotes and G allele carriers showed 3.73-fold (P=2.98×10(−8)) and 1.85-fold (P=0.002) increased risk of gastric adenocarcinoma, respectively. Stratified analyses indicated that variant genotypes had a strong association with the clinic-pathological features of gastric cancer including differentiation grade, depth of tumor infiltration, and tumor node metastasis (TNM) stage (P<0.001). Luciferase reporter assay indicated that this SNP might be responsible for aberrant B7-H1 protein expression in gastric cancer by disrupting the interaction between miR-570 and B7-H1 mRNA. These results are consistent with our hypothesis and indicate that genetic polymorphisms influencing B7-H1 expression modify cancer susceptibility.

-   7. Clinical cancer research: an official journal of the American     Association for Cancer Research/15(3):971-9; Pub Date: Feb. 1, 2009;     “Overexpression of PD-L1 significantly associates with tumor     aggressiveness and postoperative recurrence in human hepatocellular     carcinoma”; Qiang G et al. The authors published the following:—

PURPOSE The aberrant expression of programmed cell death 1 ligands 1 and 2 (PD-Ls) on tumor cells dampens antitumor immunity, resulting in tumor immune evasion. In this study, we investigated the expression of PD-Ls in human hepatocellular carcinoma (HCC) to define their prognostic significance after curative surgery.

EXPERIMENTAL DESIGN Immunohistochemistry was used to investigate PD-Ls expression as well as granzyme B+ cytotoxic and FoxP3+ regulatory T cell infiltration on tissue microarrays containing 240 randomly selected HCC patients who underwent surgery. The results were further verified in an independent cohort of 125 HCC patients. PD-Ls expression on HCC cell lines was detected by Western blot assay.

RESULTS Patients with higher expression of PD-L1 had a significantly poorer prognosis than patients with lower expression. Although patients with higher expression of PD-L2 also had a poorer survival, the difference in recurrence was not statistically significant. Multivariate analysis identified tumor expression of PD-L1 as an independent predictor for postoperative recurrence. No correlation was found between PD-Ls expression and granzyme B+ lymphocyte infiltration, whereas a significant positive correlation was detected between PD-Ls expression and FoxP3+ lymphocyte infiltration. In addition, tumor-infiltrating cytotoxic and regulatory T cells were also independent prognosticators for both survival and recurrence. The prognostic value of PD-L1 expression was validated in the independent data set.

CONCLUSION Our data suggest for the first time that PD-L1 status may be a new predictor of recurrence for HCC patients and provide the rationale for developing a novel therapy of targeting the PD-L1/PD-1 pathway against this fatal malignancy.

Using this analysis, the inventor has devised the following aspects of the invention that are useful for addressing PD-L1-mediated diseases and conditions, such as cancer, autoimmune or inflammatory diseases and conditions as more fully described below. Thus, the invention provides the following aspects.

In an example, the invention provides a method of treating or reducing the risk of a PD-L1-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human PD-L1 protein. The invention also provides a corresponding ligand. In an embodiment, the PD-L1 is encoded by a PD-L1 nucleotide sequence comprising a variation set out in Table 21, eg, comprising one or more of said SNPs (eg, RS1411262 and/or rs4143815; or 8923C and/or 395C). In a preferred embodiment, the variation is 8923C.

The present invention provides anti-PD-L1 ligands; and PD-L1-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of PD-L1, in particular human PD-L1 or its ligands and in screening assays to identify other antagonists of PD-L1 activity. Some of the ligands of the invention are useful for inhibiting PD-L1-mediated activities.

Anti-PD-L1 ligands (eg, antibodies and anti-sense RNA) have been developed based on targeting and neutralising so-called “wild-type” human PD-L1, which is a commonly-occurring form. While such therapies are useful for human patients harbouring this form of human PD-L1, the inventor considered it useful to investigate the possibility of targeting rarer—but still naturally-occurring—forms of PD-L1 amongst human populations. In this way, the inventor arrived at insight into the natural occurrences and distributions of rarer human PD-L1 forms that can serve as useful targets (at the protein or nucleic acid level) for human treatment, prophylaxis and diagnosis pertinent to diseases and conditions mediated or associated with PD-L1 activity. This particularly provides for tailored therapies, prophylaxis and diagnosis in humans that are devoid of the common PD-L1 gene or protein.

The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in activity and/or conformation of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to more effectively tailor medicines and diagnosis of patients. The invention, therefore, provides for tailored pharmaceuticals and testing that specifically addresses rarer PD-L1 polymorphic variant forms. Such forms or “alleles” (at the nucleotide level), comprise one or more changes at the nucleotide and amino acid levels from the corresponding common form nucleotide and amino acids sequences, ie, there are one or more non-synonymous (aka “missense”) changes at the nucleotide level that translate into one or more corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting such rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention. In particular, human PD-L1 variations are correlated with increased incidence or risk of PD-L1 mediated diseases or conditions, such as cancer. The inventor analysed such variations and devised the collections of variants in Table 21. In this respect, therefore, the invention provides various aspects and aspects as set out below in this example.

The inventor saw that there is significant industrial and medical application for the invention in terms of guiding the choice of anti-TOI (PD-L1) ligand for administration to human patients for therapy and/or prophylaxis of PD-L1-mediated or associated diseases or conditions. In this way, the patient receives drugs and ligands that are tailored to their needs—as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

In developing this thinking, in a non-limiting embodiment the present inventor decided to determine a set of human PD-L1 variants on the basis of the following criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population. In an embodiment the inventor selected variants having at least 3 of the 4 following criteria:—

-   -   Naturally-occurring human TOI variation having a cumulative         human allele frequency of 49 or 35% or less;     -   Naturally-occurring human TOI variation having a total human         genotype frequency of about 50% or less;     -   Naturally-occurring human TOI variation found in many different         human ethnic populations (using the standard categorisation of         the 1000 Genomes Project; see Table 2 below); and     -   Naturally-occurring human TOI variation found in many         individuals distributed across such many different ethnic         populations.

In an example, the ligand of the invention comprises an anti-human PD-L1 binding site, wherein the binding site is a human or humanized binding site, eg, the binding site comprises or consists of a human or humanized antibody variable domain or plurality of variable domains (eg, human VH/VL binding site(s)). Additionally or alternatively, the ligand comprises one or more human antibody constant regions (eg, a human antibody CH1, CH2, CH3 (or all of these) or Fc). In an example, the ligand is an antibody that comprises human or humanized variable regions and human constant regions (eg, bearing one or more mutations to enhance or dampen Fc function in a human patient).

In an example, the invention provides a method of targeting PD-L1 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human PD-L1 protein that is encoded by a human PD-L1 nucleotide comprising a PD-L1 variation as described herein. In an example, the human is suffering from or at risk of a PD-L1-mediated disease or condition. In an example, the method treats or reduces the risk of a PD-L1-mediated disease or condition in the human.

In an embodiment, (i) the antibody or fragment comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40.

Preferably, additionally or alternatively in an embodiment the anti-PDL-1 ligand (eg, antibody or fragment) comprises a gamma-1 constant region (eg, an IGHG1*01 constant region) as described herein.

Additionally or alternatively, in an embodiment, (i) the antibody or fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73.

In a specific embodiment, the anti-PD-L1 ligand, antibody or fragment of present invention comprises an Fc region, wherein the Fc region comprises at least one non-native amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may comprise additional and/or alternative non-native amino acid residues known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

The ligand, antibody or fragment according to the invention (eg, an anti-PD-L1 antibody or fragment) is for treating or preventing or reducing the risk of (or treats or prevents or reduces the risk of), for example, any disease or condition disclosed in U.S. Pat. No. 8,507,663, WO2011066389, WO2010036959, WO 2010077634, WO 03099196, WO 2013181634, WO2007005874, US20090317368 or U.S. Pat. No. 7,635,757, the disclosure of which diseases and conditions are incorporated herein by reference for potential inclusion in one or more aspects herein. Guidance on obtaining and testing antibodies can also be found in these publications. Suitable anti-PD-L1 ligands, antibodies or fragments for use in the invention are disclosed in these publications, the disclosure of which (including sequences thereof) are incorporated herein by reference for potential inclusion in one or more aspects herein.

Further encompassed by the invention is the use of the anti-PD-L1 ligand, antibody or fragment in the manufacture of a medicament for use to attenuate or inhibit a PD-L1-mediated disease or disorder in a human. PD-L1-mediated or related disorders which are treated by the ligand, antibody or fragment of the invention include, for example, described below.

Thus, a ligand, antibody or fragment of the invention is useful as a therapeutic agent in the treatment of a condition involving PD-L1 expression and/or activity. One embodiment, among others, is a method of treatment comprising administering an effective amount of a ligand, antibody or fragment of the invention to a patient in need thereof, wherein functional consequences of PD-L1 activation are decreased. Another embodiment, among others, is a method of treatment comprising (i) identifying a patient demonstrating PD-L1 expression or activity, and (ii) administering an effective amount of a ligand, antibody or fragment of the invention to the patient, wherein a functional consequence of PD-L1 activation are attenuated. An effective amount according to the invention is an amount that modulates (e.g. decreases) the functional consequences of PD-L1 activation so as to modulate (e.g. decrease or lessen) the severity of at least one symptom of the particular disease or disorder being treated, but not necessarily cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, comprising administering to a patient in need thereof an effective amount of one or more ligands, antibodies or fragments of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein such that the severity of at least one symptom of any of the disorders is reduced. Another embodiment of the invention, among others, is a method of antagonizing at least one effect of PD-L1 comprising contacting with or administering an effective amount of one or more ligands, antibodies or fragments of the present invention such that said at least one effect of PD-L1 is antagonized, e.g. the ability to promote or maintain cancer, an autoimmune condition or an inflammatory condition.

Tailoring Anti-PD-L1 Ligands

As outlined herein (for example, in the context of PCSK9 in Example 1), the invention includes the possibility to tailor treatment of humans further by selecting antibody-based ligands with variable domains and/or constant domains based on gene segments found in many humans of the ethnic populations where the variant TOI forms are found to meet the selection criteria of the invention. This also applies mutatis mutandis where the TOI is human PD-L1 as in the present example. Thus, all disclosure herein relating to tailoring variable and/or constant domains apply to the present example, relating to PD-L1 and is combinable for use in one or more aspects herein.

As described in Example 1, an example is provided for ligands comprising antibody VH domains derived from recombination of human IGHV gene segments comprising selected nucleotides at positions in the HCDR1 or FW3 where there is variability in humans (ie, where SNPs occur in humans).

Further information is provided in Table 4, which shows variation at these positions, as well as the variant distributions across the 1000 Genomes Project database relating to many human populations.

In other embodiments, as explained more fully above, the invention provides for ligands which are tailored to the human recipient's genotype and/or phenotype based on alternative human VH gene segments, or on Vκ, V or constant region gene segments (see further Table 9 for representative variants).

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23*04), a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40. (ii) wherein the ligand comprises a VH domain derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment, and wherein said human comprises a IGHV3-7*01 VH gene segment or the human expresses VH domains derived from the recombination of human VH segment IGHV3-7*01, a human D gene segment and a human JH segment. (iii) wherein the ligand comprises a Vκ domain derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment, and wherein said human comprises a IGKV1-12*01 Vκ gene segment or the human expresses Vκ domains derived from the recombination of human Vκ segment IGKV1-12*01 and a human Jκ segment. (iv) wherein the ligand comprises a Vκ domain derived from the recombination of a human Vκ segment and a human Jκ segment, the human Vκ segment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36 and wherein said human comprises a Vκ gene segment encoding a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the human expresses Vκ domains that comprise a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 and wherein said human comprises a Vκ gene segment encoding a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 or the human expresses Vκ domains that comprise a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38. (v) wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4. (vi) wherein the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6. In an example, the constant region is Fe enhanced, eg, ADCC or CDC enhanced. The skilled person will know techniques (eg, known C region mutations) to enhance ADCC or CDC. (vii) wherein the ligand comprises a human kappa chain constant region that comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16 and wherein said human comprises (i) an IGKC1*01 human kappa chain constant region gene segment, or the human expresses antibodies comprising human kappa chain constant regions comprising a Val corresponding to position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16. (viii) wherein the ligand comprises a human IGLC1*01 lambda chain constant region and wherein said human comprises (i) a human IGLC1*01 lambda chain constant region gene segment, or the human expresses antibodies comprising human IGLC1*01 lambda chain constant regions. (ix) wherein the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73. (x) wherein the ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3*01) constant region gene segment and wherein said human comprises (i) said first constant region gene segment (eg, an IGHG3*01), or the human expresses antibodies comprising human gamma-3 heavy chain constant regions encoded by said first human IGHG3 (eg, IGHG3*01) constant region gene segment. (xi) wherein the ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human epsilon heavy chain constant regions encoded by said first constant region gene segment. (xii) wherein the ligand comprises a human mu heavy chain constant region encoded by a first human mu heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human mu heavy chain constant regions encoded by said first constant region gene segment. (xiii) wherein the ligand comprises a human alpha heavy chain constant region encoded by a first human alpha heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human alpha heavy chain constant regions encoded by said first constant region gene segment. (xiv) wherein the ligand comprises a human delta heavy chain constant region encoded by a first human delta heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human delta heavy chain constant regions encoded by said first constant region gene segment. (xv) wherein the ligand comprises a human kappa light chain constant region encoded by a first human kappa light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human kappa light chain constant regions encoded by said first constant region gene segment. (xvi) wherein the ligand comprises a human lambda light chain constant region encoded by a first human lambda light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human lambda light chain constant regions encoded by said first constant region gene segment.

Generally, in an example the ligand comprises a heavy chain constant region that is Fe enhanced, eg, ADCC or CDC enhanced. The skilled person will know techniques (eg, known C region mutations; defucosylation to enhance ADCC) to enhance ADCC or CDC. This applies to any of Examples 1 onwards herein.

Thus, it may be advantageous for PD-L1 ligands, for the ligand to comprise a gamma-1 or gamma-2 constant region (eg, as per the embodiments (i) to (xvi) above). For example, the ligand comprises said gamma-1 constant region, eg, an ADCC or CDC enhanced constant region meeting embodiment (v).

In an example, the ligand (eg, an antibody) is conjugated to a cytokine selected from the group consisting of IL-2, IL-12, IL-15 or IL-21. In an example, the cytokine is IL-2.

In an example, eg, for where the condition is a viral infection, the ligand (eg, antibody) comprises a gamma-4 constant region as herein defined and optionally wherein an Fe inactivated IgG4, such a IgG4(S228P).

Determination of Specific Binding of Ligands of the Invention to PD-L1 Variants

The specific binding of ligands of the invention to PD-L1 variants can be performed using the SPR method described in Example 1.

By way of example, the invention thus provides the following aspects:—

1. A method of treating or reducing the risk of a disease or condition mediated by PD-L1 in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21.

For example, there is provided a method of treating or reducing the risk of a disease or condition mediated by PD-L1 in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21, wherein said human comprises a PD-L1 nucleotide sequence comprising said selected variation.

For example, there is provided a method of treating or reducing the risk of a cancer in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21, wherein said human comprises a PD-L1 nucleotide sequence comprising said selected variation.

For example, there is provided a method of treating or reducing the risk of an autoimmune disease or condition in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21, wherein said human comprises a PD-L1 nucleotide sequence comprising said selected variation.

For example, there is provided a method of treating or reducing the risk inflamatory disease or condition in a human, the method comprising administering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to a human PD-L1 that is expressed by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21, wherein said human comprises a PD-L1 nucleotide sequence comprising said selected variation.

Herein, specific binding can be tested using a sample PD-L1 using a routine method known the skilled person, eg, ELISA or SPR (eg, using a method as described herein). For example, the PD-L1 is provided by a sample from said human or another human, eg, a serum or blood sample.

In an example, the ligand comprises an anti-PD-L1 binding site that specifically binds human PD-L1. Optionally, the binding site comprises one or more antibody variable domains or one or more human PD-L1 receptor domains. In an example, specific binding with a Kd of 1 mm or less (ie, 1 mM or stronger binding), eg, 1 nM or less; or 100 pM or less; or 10 pM or less. Specific binding can be determined, for example, by assessing binding of the ligand to PD-L1 in a sample from said human or another human comprising said PD-L1. In an example, the sample comprises serum, blood, faeces, tissue, a cell, urine and/or saliva of said human.

In an example, the ligand is selected from the group consisting of wher MED14736 (Medimmune), durvalumab, RG-7446 (Genentech, Roche, Chugai, MPDL3280A), MPDL3280A (Genentech), STI-A1014 (Sorrento Therapeutics Inc), MSB-0010718C (Merck Serono); MDX1105 (Bristol-Myers Squibb) and BMS-936559 (Bristol-Myers Squibb). Optionally the ligand is administered in combination with an anti-CTLA-4 ligand (eg, antibody or fragment), eg, Tremelimumab. For example, MEDI4736 or durvalumab is administered in combination with an anti-CTLA-4 ligand (eg, antibody or fragment), eg, Tremelimumab.

In an example, the ligand is an anti-PD-L1 antibody or antibody fragment, eg, durvalumab.

In an example, the ligand is an NCE (so-called New Chemical Entity in the art).

In an example, the human has been diagnosed with said disease or condition before said administration of the ligand.

For example, the human is partially or completely resistant to treatment with an anti-PD-L1 ligand, eg, an anti-PD-L1 NCE.

In an example, the ligand is administered by intravenous or subcutaneous administration and/or is comprised in an injectable preparation.

2. The method of aspect 1, wherein the nucleotide sequence comprises a nucleotide corresponding to SNP rS1411262, SNP rs4143815, 8923C or 395C. 3. The method of aspect 1 or 2, wherein said human comprises a PD-L1 nucleotide sequence comprising said selected variation.

Optionally, the human is homozygous for said variation. Alternatively, the human is heterozygous for said variation.

In an example, the ligand comprises an antibody constant region (eg, an antibody Fc region).

4. The method of any one of aspects 1 to 3, wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an amino acid selected from the group consisting of an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and optionally wherein said human comprises an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising said selected amino acid.

For example, the ligand comprises a human gamma-I heavy chain constant region that comprises an amino acid selected from the group consisting of an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising said selected amino acid. In an embodiment, this is combined with any of the examples set out under aspect 1 above.

Optionally, this constant region of the ligand is comprised by an Fc region, eg, an enhanced Fc region.

Optionally, the ligand comprises an IGHG1*01 human heavy chain constant region.

5. The method of any one of aspects I to 4, wherein the disease or condition is cancer (eg, a solid tumour), an autoimmune disease or condition; or an inflammatory disease or condition. In an example, the cancer is selected from the group consisting of breast cancer, lung cancer (eg, non-small cell lung cancer), colon cancer, ovarian cancer, melanoma (eg, stage IV melanoma), bladder cancer, liver cancer (eg, hepatocellular carcinoma), kidney cancer, salivary gland cancer, stomach cancer, gastric cancer, glioma, pancreas cancer, thyroid cancer, thymic epithelial cancer, head cancer and/or neck cancer, multiple myeloma, T cell lymphoma, Hodgkin lymphoma, renal cell carcinoma, cervical cancer, colorectal, a haematological neoplasm, metastatic colorectal cancer, uterine or cervix cancer, a leukaemia (eg, acute myelogenous leukaemia), diffuse large B-cell lymphoma, follicle centre lymphoma, prostate cancer and Merkel cell carcinoma.

Alternatively, the disease or condition is a viral infection, eg, HIV, hepatitis C or Ebola infection. Alternatively, the disease or condition is melodysplastic syndrome.

In an example, the condition is a cancer, eg, metastatic cancer.

6. The method of any one of aspects 1 to 5, comprising, before said administering, selecting a human comprising said PD-L1 nucleotide sequence, wherein the human is the human of aspect 1. 7. The method of any one of aspects 1 to 6, wherein the human has been determined to comprise said PD-L1 nucleotide sequence. 8. The method of any one of aspects 1 to 6, comprising the step of determining that the human comprises said PD-L1 nucleotide sequence, optionally, wherein the determining step is performed before administration of the antibody to the human. 9. The method of aspect 8, wherein the step of determining comprises assaying a biological sample from the human for a PD-L1 nucleotide sequence comprising said selected variation. 10. The method of aspect 9, wherein the assaying comprises contacting the biological sample with

-   -   a. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence comprising said selected variation or that specifically         hybridizes to an antisense of said sequence, wherein said         nucleic acid hybridizes to said selected variation or hybridizes         to an antisense sequence thereby forming a complex when at least         one nucleotide sequence comprising said selected variation is         present; and/or     -   b. at least one oligonucleotide probe comprising a sequence of         at least 10 contiguous nucleotides of a nucleotide sequence         comprising said selected variation or comprising an antisense         sequence of said contiguous nucleotides, wherein said sequence         of contiguous nucleotides comprises said selected variation         thereby forming a complex when the nucleotide sequence         comprising said selected variation is present; and detecting the         presence or absence of the complex, wherein detecting the         presence of the complex determines that the human comprises a         PD-L1 nucleotide sequence comprising said selected variation.         11. The method of aspect 10, wherein the assaying comprises         nucleic acid amplification and optionally one or more methods         selected from sequencing, next generation sequencing, nucleic         acid hybridization, and allele-specific amplification and/or         wherein the assaying is performed in a multiplex format.         12. The method of any one of aspects 1 to 11, wherein said human         is or has been further determined to be substantially resistant         a PD-L1 or PD-1 treatment.         13. The method of any one of aspects 1 to 11, wherein said human         is receiving or has received a PD-L1 or PD-1 treatment or has         reduced responsiveness to a PD-L1 or PD-1 treatment.         14. The method of aspect 9, wherein said biological sample         comprises serum, blood, faeces, tissue, a cell, urine and/or         saliva of said human.         15. The method of any one of aspects 1 to 14, wherein said human         has been diagnosed with a cancer, autoimmune disease or         condition; or an inflammatory disease or condition.         16. The method of any one of aspects 1 to 15, wherein said         ligand is administered by intravenous or subcutaneous injection         and/or is comprised in an injectable preparation.         17. The method of any one of aspects 1 to 16, wherein the ligand         is human.         18. An anti-human PD-L1 ligand (eg, an antibody, antibody         fragment or human PD-L1 trap) for use in a method of any one of         aspects 1 to 17.         19. The ligand of aspect 18, wherein the ligand is a human         anti-human PD-L1 antibody (eg, durvalumab), antibody fragment or         human PD-L1 trap.         20. The ligand of aspect 18 or 19, wherein said ligand is for         intravenous or subcutaneous administration and/or is comprised         in an injectable preparation.

For rs148170925, the 1000 Genomes Phase 1 database indicates a cumulative frequency across all human populations of 60% for the deletion (ie, presence on the SNP) and 40% for absence of the SNP (ie, presence of TG at chromosomal location 9:5454642-5454643). The following human populations have above average frequency for absence of the SNP: AMR (45%), EUR (54%), ASW (44%), CLM (42%), MXL (44%), PUR (49%), CEU (60%), FIN (57%), GBR (56%), IBS (46%) and TSI (46%); in an embodiment of the invention, the human is of any one of these ancestries. The following human populations have above average frequency for presence of the SNP: AFR (67%), ASN (76%), LWK (77%), YRI (65%), CHB (73%), CHS (79%), JPT (75%); in an embodiment of the invention, the human is of any one of these ancestries.

Example 7: TOIs For Cancer, Autoimmune & Inflammatory Applications Example 7A: Tumour Targeting & Immuno-Oncology Applications

The tumour microenvironment is an important aspect of cancer biology that contributes to tumour initiation, tumour progression and responses to therapy. Cells and molecules of the immune system are a fundamental component of the tumour microenvironment. Importantly, therapeutic strategies can harness the immune system to specifically target tumour cells and this is particularly appealing owing to the possibility of inducing tumour-specific immunological memory, which might cause long-lasting regression and prevent relapse in cancer patients.

The composition and characteristics of the tumour microenvironment vary widely and are important in determining the anti-tumour immune response. For example, certain cells of the immune system, including natural killer cells, dendritic cells (DCs) and effector T cells, are capable of driving potent anti-tumour responses. However, tumour cells often induce an immunosuppressive microenvironment, which favours the development of immunosuppressive populations of immune cells, such as myeloid-derived suppressor cells and regulatory T cells. Understanding the complexity of immunomodulation by tumours is important for the development of immunotherapy. Various strategies are being developed to enhance anti-tumour immune responses, including DC-based vaccines and antagonists of inhibitory signalling pathways to overcome ‘immune checkpoints’.

The invention provides methods involving administering an anti-TOI ligand to a human, the TOI being present in humans as a plurality of variants differing by one or more amino acid polymorphisms, the method comprising administering a ligand to the human, the ligand comprising first and second protein domains, wherein the first domain specifically binds a TOI variant comprising a first amino acid polymorphism, wherein the second domain comprises a second polymorphism, and wherein the human expresses (i) TOI comprising said first amino acid polymorphism; and (ii) protein domains comprising said second polymorphism.

Particularly, the invention provides a method of targeting a tumour in a human and, eg, in this case the TOI is a tumour antigen (eg, a tumour-specific antigen or a tumour-associated antigen, as will be clear to the skilled addressee). For example, the TOI is selected from the group consisting of human PD-L1, Alphafetoprotein (AFP), Carcinoembryonic antigen (CEA), CA-125, MUC-1, Epithelial tumor antigen (ETA) and Melanoma-associated antigen (MAGE).

Optionally the ligand comprises a human cytokine variant amino acid sequence, optionally the cytokine being selected from the group consisting of IL-2, IL-12, IL-15 and IL-21, eg, wherein the sequence is fused to the N-terminus of one or more heavy and/or light chains of the ligand when the ligand is an antibody or fragment or a trap comprising Fc regions.

In an example, the cytokine is human IL-2 and the sequence comprises a polymorphism selected from 1421, 129D, 76L, 38L and 27T. For example, the sequence comprises 1421 and 129D. These positions are indicated to be polymorphic and associated with SNPs in Ensembl and dbSNP. In an embodiment, the ligand comprises an IL-2 amino acid sequence comprising one, more or all of the polymorphisms of said group. In an example, the cytokine comprises SEQ ID NO: 134.

In an example, the cytokine is human IL-21 and the sequence comprises a polymorphism selected from 32Q, 40R, 431, 98V, 106K, 112A, 113G, 134K and 135E. For example, the sequence comprises 32Q and 40R. These positions are indicated to be polymorphic and associated with SNPs in Ensembl and dbSNP. In an embodiment, the ligand comprises an IL-21 amino acid sequence comprising one, more or all of the polymorphisms of said group.

In an alternative, there is provided a method of cancer immunotherapy and, eg, in this case the TOI is a human immune cell antigen (eg, wherein the antigen is a human natural killer cell, dendritic cell (DC) or effector T cell antigen). In another example the TOI is an immune checkpoint target.

In an embodiment the TOI is human PD-1. In an embodiment the TOI is human PD-L1 or PD-L2. In another embodiment the target is human CTLA-4. In another embodiment the target is human LAG-3. In another embodiment the target is human TIM-3. In an embodiment the TOI is human OX40 (and optionally the ligand agonises the TOI). In an embodiment the TOI is human KIR. In an embodiment the TOI is human CD137 (and optionally the ligand agonises the TOI). In an embodiment the TOI is human CD27 (and optionally the ligand agonises the TOI). In an embodiment the TOI is human CD70. In an embodiment the TOI is human B7-H3. In an embodiment the TOI is human KIR. In an embodiment the TOI is human GITR (and optionally the ligand agonises the TOI). In an example, the ligand comprises a human cytokine variant amino acid sequence as described above, the cytokine being useful for immunotherapy of cancer. Where an agonist is not mentioned, the ligand can for example antagonise the TOI.

In an example the ligand is selected from the ligands of Table 22.

TABLE 22 Ligands ipilimumab nivolumab pembrolizumab durvalumab tremelimumab RG-7446 lirilumab MEDI-6469 MSB-0010718C pidilizumab AMP-514 TRX-518 BMS-986016 urelumab varlilumab PF-05082566 SGN-CD70A ARGX-110 MGA-271 rHIgM12B7

In an example of either alternative the method treats or reduces the risk of a cancer, eg, any cancer disclosed herein (eg, as disclosed in Example 5, 6 or this Example 7).

Thus, an alternative provides: A method of cancer immunotherapy in a human by targeting an immune cell TOI in the human, the TOI being present in humans as a plurality of variants differing by one or more amino acid polymorphisms, the method comprising administering a ligand to the human, the ligand comprising first and second protein domains, wherein the first domain specifically binds a TOI variant comprising a first amino acid polymorphism, wherein the second domain comprises a second polymorphism, and wherein the human expresses (i) TOI comprising said first amino acid polymorphism; and (ii) protein domains comprising said second polymorphism.

The TOI is for example PD-1 (also known as PDCD1, programmed cell death 1, PD1 and CD279), CTLA-4, LAG-3 or TIM-3. For example, the TOI is human PD-1 and the first polymorphism is a PD-1 polymorphism described herein (eg, a variation listed in Table 23). For example the polymorphism is encoded by a PD-1 SNP described herein.

It may be desirable to block (not necessarily clear as with an IgG1 format) the TOI in the method of this alternative and a human IgG4 may be preferable for doing this in a cancer immunotherapy setting. Thus, in an embodiment, the ligand comprises a human gamma-4 constant region, wherein the constant region comprises said second domain. Importantly, according to the invention, the constant region polymorphism is matched with the genome of the human, thereby minimising interfering immune responses, as may be desirable to minimise in the current cancer immunotherapy setting. “Blocking” of the TOI means, for example, antagonising the binding of the TOI to a cognate receptor or human biological target, eg, blocking PD-1 binding to PD-L1 and/or PD-L2; CTLA-4 binding to B7-1 or B7-2; or CD28 binding to B7-1 or B7-2.

For the cancer immunotherapy setting of the invention, it may be advantageous wherein the gamma-4 is a gamma-4 constant region with 228Pro or 235Glu. In an example the constant region is an IgG4PE (ie, a gamma-4 constant region with 228Pro and 235Glu). In an example, the ligand comprises human IGHG4*01 hinge S10>P constant region.

In an example, therefore, the ligand comprises any human gamma-4 constant region disclosed herein (eg, in any of Examples 1 et seq).

The present invention, thus, provides the following concepts:—

-   -   1. A method of cancer immunotherapy in a human by targeting an         immune cell TOI (eg, PD-1) in the human, the TOI being present         in humans as a plurality of variants differing by one or more         amino acid polymorphisms, the method comprising administering a         ligand (eg, an antibody or antibody fragment) to the human, the         ligand comprising first and second protein domains, wherein the         first domain specifically binds a TOI variant comprising a first         amino acid polymorphism, wherein the second domain comprises a         second polymorphism, and wherein the human expresses (i) TOI         comprising said first amino acid polymorphism; and (ii) protein         domains comprising said second polymorphism, wherein the ligand         comprises a human gamma-4 heavy chain constant region that         comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg         at position 289 shown in SEQ ID NO: 73 and wherein said human         comprises an IGHG4*01 human heavy chain constant region gene         segment, or the human expresses antibodies comprising human         gamma-4 heavy chain constant regions comprising a Leu at         position 189 shown in SEQ ID NO: 73 or an Arg at position 289         shown in SEQ ID NO: 73; and wherein the second domain is         comprised by said gamma-4 heavy chain constant region of the         ligand.

Optionally, the TOI is a human immune cell antigen (eg, wherein the antigen is a human natural killer cell, dendritic cell (DC) or effector T cell antigen).

The programmed death-1 (PD-1) receptor serves as an immunologic checkpoint, limiting bystander tissue damage and preventing the development of autoimmunity during inflammatory responses. PD-1 is expressed by activated T cells and down-modulates T-cell effector functions upon binding to its ligands, PD-L1 and PD-L2, on antigen-presenting cells. In patients with cancer, the expression of PD-1 on tumor-infiltrating lymphocytes and its interaction with the ligands on tumor and immune cells in the tumor microenvironment undermines antitumor immunity and supports the rationale for PD-1 blockade in cancer immunotherapy. In a particularly preferred embodiment, there is provided a method of cancer immunotherapy in a human by targeting PD-1 in the human.

-   -   2. The method of concept 1, wherein the first and/or second         polymorphism is respectively encoded by a SNP having a         cumulative human allele frequency of less than 50%.

Optionally the frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

-   -   3. The method of any preceding concept, wherein the first domain         is an antibody variable domain or a receptor domain.     -   4. The method of any preceding concept, wherein the second         domain is an antibody domain, optionally an antibody variable or         constant domain.     -   5. The method of any preceding concept, wherein the second         domain is a domain of an antibody Fc region.     -   6. The method of any one of concepts 1 to 4, wherein the first         and second domains are antibody variable domains, optionally         wherein the variable domains specifically bind first and second         human TOI respectively, wherein the TOIs are different.     -   7. The method of any one of concepts 1 to 4, wherein the first         and second domains are receptor domains, optionally wherein the         receptor domains specifically bind first and second human TOI         respectively, wherein the TOIs are different.     -   8. The method of concept 6 or 7, wherein the first domain         comprises a third amino acid polymorphism, wherein the human         expresses (iii) domains comprising said third amino acid         polymorphism.

Optionally the human expresses the second TOI variant. In an example, the second TO is a human immune cell surface antigen or a tumour antigen.

Optionally the third polymorphism is encoded by a SNP having a cumulative human frequency of less than 50%. Optionally the frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or I %.

In an example, the first domain is an antibody variable domain (eg, any V domain of the invention described herein, such human VH3-23*04 as herein described) and the domains of (iii) are antibody variable domains. For example, the first domains and domains of (iii) are VH domains. For example, the first domains and domains of (iii) are Vκ domains. For example, the first domains and domains of (iii) are VW domains.

In an example, the first domain is a receptor binding site and the domains of (iii) are said receptor binding site domains.

-   -   9. A method according to concepts 1, 2 and 8 combined.     -   10. The method of any one of concepts 1 to 3, wherein the second         domain is a domain of a human cytokine variant, optionally         selected from the group consisting of IL-2, IL-12, IL-15 and         IL-21, and the domains of (ii) are domains of said cytokine         variant.

In an example, the second domain is a domain of human IL-2 and the second polymorphism is selected from the group consisting of 1421, 129D, 76L, 38L and 27T. For example, the second domain comprises 1421 and 129D. These positions are indicated to be polymorphic and associated with SNPs in Ensembl and dbSNP. In an embodiment, the ligand comprises an IL-2 amino acid sequence comprising one, more or all of the polymorphisms of said group.

In an example, the second domain is a domain of human IL-21 and the second polymorphism is selected from the group consisting of 32Q, 40R, 431, 98V, 106K, 112A, 113G, 134K and 135E. For example, the second domain comprises 32Q and 40R. These positions are indicated to be polymorphic and associated with SNPs in Ensembl and dbSNP. In an embodiment, the ligand comprises an IL-21 amino acid sequence comprising one, more or all of the polymorphisms of said group.

-   -   11. The method of any preceding concept, wherein the human         expresses the first and/or second domain.     -   12. The method of any preceding concept, wherein the first,         second and/or third polymorphism is respectively found in at         least 5 different human populations as defined in Table 4.

For example, each of the first and second polymorphisms is respectively found in at least 5 different human populations as defined in Table 4. For example, each of the first, second and polymorphisms is respectively found in at least 5 different human populations as defined in Table 4.

Optionally the first, second and/or third (see below) polymorphism respectively appears in at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different human populations as defined in Table 4.

-   -   13. The method of concept 12, wherein the first, second and/or         third polymorphism respectively appears in at least 15 humans in         the 1000 Genomes Phase I database.

In an embodiment, the first, second and/or third polymorphism is found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 human individuals distributed across such many different human populations.

-   -   14. The method of any preceding concept, wherein the first,         second and/or third polymorphism is respectively found in at         least 5 different human populations as defined in Table 4 and is         encoded by a SNP having a cumulative human allele frequency of         less than 50%.

Optionally the frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

-   -   15. The method of any preceding concept, wherein the first,         second and/or third polymorphism is respectively encoded by a         SNP having total human genotype frequency of less than 50%.

Optionally the frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

-   -   16. The method of any preceding concept, wherein the first and         second domains are human, optionally wherein the ligand is fully         human.     -   17. The method of any preceding concept, wherein the ligand is         an anti-TOI trap, antibody or antibody fragment.     -   18. The method of any preceding concept, wherein the ligand         specifically binds two or more human TOIs, eg, said first and         second TOIs.     -   19. The method of any preceding concept, wherein the ligand is         administered to the human by injection or the ligand is provided         by an injectable preparation.     -   20. The method of any preceding concept, wherein said human has         been or is genotyped as positive for said TOI variant nucleotide         sequence before administering the ligand, or the method         comprises genotyping the human as positive for said variant         nucleotide sequence before administering the ligand.     -   21. The method of any preceding concept, wherein the human has         been or is phenotyped as positive for said TOI variant before         administering the ligand, or the method comprises phenotyping         the human as positive for said variant before administering the         ligand.     -   22. The method of any preceding concept, comprising, before said         administering, selecting a human comprising said first TOI         polymorphism, wherein the human is the human of concept 1.     -   23. The method of any preceding concept, wherein the human has         been determined to comprise the first and/or second TOI variant         or a nucleotide sequence encoding the TOI variant(s).     -   24. The method of any preceding concept, comprising the step of         determining that the human comprises a first TOI nucleotide         sequence encoding the first polymorphism, optionally, wherein         the determining step is performed before administration of the         ligand to the human.     -   25. The method of concept 24 wherein the step of determining         comprises assaying a biological sample from the human for said         nucleotide sequence.     -   26. The method of concept 25, wherein the assaying comprises         contacting the biological sample with         -   a. a. at least one oligonucleotide probe comprising a             sequence of at least 10 contiguous nucleotides that can             specifically hybridize to and identify in the biological             sample a nucleotide sequence comprising a SNP encoding the             first polymorphism or that specifically hybridizes to an             antisense of said sequence, wherein said nucleic acid             hybridizes to said SNP or hybridizes to an antisense             sequence thereby forming a complex when at least one             nucleotide sequence comprising said SNP is present; and/or         -   b. b. at least one oligonucleotide probe comprising a             sequence of at least 10 contiguous nucleotides of a             nucleotide sequence comprising said SNP or comprising an             antisense sequence of said contiguous nucleotides, wherein             said sequence of contiguous nucleotides comprises said SNP             thereby forming a complex when the nucleotide sequence             comprising said SNP is present; and         -   c. detecting the presence or absence of the complex, wherein             detecting the presence of the complex determines that the             human comprises the first TOI nucleotide sequence.     -   27. The method of concept 25, wherein the assaying comprises         nucleic acid amplification and optionally one or more methods         selected from sequencing, next generation sequencing, nucleic         acid hybridization, and allele-specific amplification and/or         wherein the assaying is performed in a multiplex format.     -   28. The method of any preceding concept, wherein said human is         or has been further determined to be substantially resistant to         a cancer treatment.     -   29. The method of any preceding concept, wherein said human is         receiving or has received cancer treatment or has reduced         responsiveness to a cancer treatment.     -   30. The method of concept 25, wherein said biological sample         comprises serum, blood, faeces, tissue, a cell, urine and/or         saliva of said human.     -   31. The method of any preceding concept, wherein said human has         been diagnosed with said disease or condition.     -   32. An anti-TOI ligand (eg, an antibody, antibody fragment or         trap) for use in the method of any preceding concept. For         example, the TOI is human PD-1 (eg, the ligand is is         pembrolizumab, lambrolizumab or KEYTRUDA™. In an example, the         ligand is nivolumab or OPDIVO™). For example, the TOI is human         CTLA-4, eg, the ligand is ipilimumab, YERVOY™, tremelimumab,         ticilimumab, belatacept or Nulojix™.

In an embodiment, the trap is a receptor Fc fusion (ie, a receptor domain fused to an antibody Fc region, eg, as a dimer), wherein the receptor specifically binds to the TOI.

-   -   33. The ligand of concept 32, wherein the ligand is fully human.     -   34. The ligand of concept 32 or 33, wherein the ligand is for         administration by injection or the ligand is provided by an         injectable preparation; wherein the ligand is for topical or         inhaled administration or the ligand is provided by a topical or         inhalable preparation.

In an example, the first, second and/or third polymorphism respectively:—

-   -   d. has a cumulative human allele frequency of 15% or less;     -   e. has a total human genotype frequency of 20% or less;     -   f. is found in at least 5 different human populations (using the         standard categorisation of the 1000 Genomes Project, eg, as per         the Phase I database); and     -   g. is found in many individuals distributed across such many         different populations.

In an example, the criteria are applied with reference to one or more human genomic sequence databases as described herein. For example, the criteria are those as applied to the 1000 Genomes database.

For example in any aspect example, embodiment or configuration of the invention, the 1000 Genomes database release 13 or Phase I. For example, the 1000 Genomes database in its most recent version as at 1 Oct. 2013 or 1 Oct. 2014.

In an example, the human is homozygous for the first polymorphism. Additionally or alternatively, the human is homozygous for the first polymorphism. Additionally or alternatively, the human is homozygous for the third polymorphism. For example, the human is homozygous for the first and second polymorphisms.

In an example, the method is according to any one of the disclosure herein involving selecting, genotyping or phenotyping the human as positive for the TOI variant (ie, in the present case human PD-1 comprising the polymorphism or a nucleotide sequence encoding this). In an example, the method is according to any one of concepts above involving the determining step or wherein the human has been determined to comprise the variant TOI variant (eg, in the present case human PD-1 comprising the polymorphism or a nucleotide sequence encoding this).

In an example, the first polymorphism is a human PD-1 amino acid polymorphism described herein or is encoded by a human PD-1 SNP described herein.

In an example, wherein the TOI is human PD-1 the first polymorphism is encoded by a SNP selected from the group consisting of rs7568402, rs28699177, rs28542728, rs118117097, rs28570544, rs118027315, rs191919871, rs6707556 and rs142909968.

The invention further provides the following specific aspects:—

-   1. A method of cancer immunotherapy in a human by targeting PD-1 in     the human, the method comprising administering an antibody or     antibody fragment to the human, wherein the antibody or antibody     fragment specifically binds a PD-1 comprising a first amino acid     polymorphism, wherein the antibody or antibody fragment comprises a     human gamma-4 heavy chain constant region that comprises a Leu at     position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown     in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01     human heavy chain constant region gene segment, or the human     expresses antibodies comprising human gamma-4 heavy chain constant     regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or     an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a PD-1     nucleotide sequence encoding a PD-1 comprising said first amino acid     polymorphism or the human expresses a PD-1 comprising said first     amino acid polymorphism.

A first alternative aspect 1 provides:—

A method of cancer immunotherapy in a human by targeting PD-1 in the human, the method comprising administering an antibody or antibody fragment to the human, wherein the antibody or antibody fragment specifically binds a PD-1 encoded by a PD-1 nucleotide sequence comprising a SNP (first polymorphism) selected from the group consisting of the variations set out in Table 23 (eg, selected from the group consisting of rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982), wherein the antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide sequence comprising said selected SNP.

These SNPs are discussed further below.

A first alternative aspect 1 provides:—

A method of treating cancer in a human, the method comprising administering an antibody or antibody fragment to the human, wherein the antibody or antibody fragment inhibits the binding of PD-L1 or PD-L2 to a PD-1 comprising a first amino acid polymorphism, wherein the antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide sequence encoding a PD-1 comprising said first amino acid polymorphism or the human expresses a PD-1 comprising said first amino acid polymorphism. In an example, the human comprises a PD-1 nucleotide sequence comprising a SNP selected from the group consisting of a PD-L1 SNP disclosed in Example 6, or PD-1 SNP rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982; optionally wherein the human has been determined to comprise said SNP or the method comprises determining that the human comprises said SNP before administering the antibody or fragment.

In an example the antibody or fragment specifically binds human PD-L1, PD-L2 or PD-1. In an example the antibody or fragment specifically binds human PD-L1. In an example the antibody or fragment specifically binds human PD-1.

For example, the PD-L1 comprises a PD-L1 polymorphism described in Example 6 or is encoded by a PD-L1 nucleotide sequence comprising a PD-L1 SNP described in Example 6.

For example, the PD-1 comprises a PD-1 polymorphism described herein (eg, in Table 23) or is encoded by a PD-1 nucleotide sequence comprising a PD-1 SNP described herein (eg, in Table 23), eg, a SNP selected from the group consisting of rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982. These SNPs are discussed further below.

-   2. The method of aspect 1, wherein the first polymorphism or     selected SNP is a nucleotide corresponding to SNP rs36084323,     rs10204225, rs11568821, rs2227981 or rs2227982. -   3. The method of aspect 1 or 2, wherein said human is homozygous for     said first polymorphism or selected SNP. -   4. The method of any preceding aspect, wherein the ligand comprises     an IGHG4*01 human heavy chain constant region. -   5. The method of any preceding aspect, wherein the cancer is     selected from the group consisting of renal cell carcinoma;     haematological neoplasm; stage IV melanoma; non small-cell lung     cancer; metastatic stomach cancer; breast cancer; solid tumour;     bladder cancer; head and neck cancer, colorectal cancer,     glioblastoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's     lymphoma, follicular lymphoma, cervical cancer, myeloid leukaemia     (eg, acute myeloid leukaemia or chronic myeloid leukaemia). In an     embodiment, the cancer is not melanoma. In an embodiment, the cancer     is not lung cancer. In an embodiment, the cancer is not non     small-cell lung cancer. -   6. The method of any preceding aspect, comprising, before said     administering, selecting a human comprising said PD-1 nucleotide     sequence of (ii), wherein the human is the human of aspect 1. -   7. The method of any preceding aspect, wherein the human has been     determined to comprise said PD-L nucleotide sequence of (ii). -   8. The method of any preceding aspect, comprising the step of     determining that the human comprises said PD-L nucleotide sequence     of (ii), optionally, wherein the determining step is performed     before administration of the antibody to the human. -   9. The method of aspect 8, wherein the step of determining comprises     assaying a biological sample from the human for a PD-L nucleotide     sequence comprising said selected variation. -   10. The method of aspect 9, wherein the assaying comprises     contacting the biological sample with     -   a. a. at least one oligonucleotide probe comprising a sequence         of at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence comprising said selected variation or that specifically         hybridizes to an antisense of said sequence, wherein said         nucleic acid hybridizes to said selected variation or hybridizes         to an antisense sequence thereby forming a complex when at least         one nucleotide sequence comprising said selected variation is         present; and/or     -   b. b. at least one oligonucleotide probe comprising a sequence         of at least 10 contiguous nucleotides of a nucleotide sequence         comprising said selected variation or comprising an antisense         sequence of said contiguous nucleotides, wherein said sequence         of contiguous nucleotides comprises said selected variation         thereby forming a complex when the nucleotide sequence         comprising said selected variation is present; and     -   c. detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises a PD-L1 nucleotide sequence comprising said selected         variation. -   11. The method of aspect 10, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of any preceding aspect, wherein said human is or has     been further determined to be substantially resistant a cancer,     PD-L1 or PD-1 treatment.

For example the human is or has been further determined to be resistant to a chemotherapy treatment.

-   13. The method of any preceding aspect, wherein said human is     receiving or has received a cancer (eg, chemotherapy), PD-L1 or PD-1     treatment or has reduced responsiveness to a cancer (eg,     chemotherapy), PD-L1 or PD-1 treatment. -   14. The method of aspect 9, wherein said biological sample comprises     serum, blood, faeces, tissue, a cell, urine and/or saliva of said     human. -   15. The method of any preceding aspect, wherein said ligand is     administered by intravenous or subcutaneous injection and/or is     comprised in an injectable preparation. -   16. The method of any preceding aspect, wherein the ligand is human,     eg, a human antibody or antibody fragment. -   17. An anti-human PD-1 ligand (eg, an antibody, antibody fragment or     trap) for use in the method of any preceding aspect.

For example, the ligand is is pembrolizumab, lambrolizumab or KEYTRUDA™. In an example, the ligand is nivolumab or OPDIVO™).

In an embodiment, the trap is a receptor Fc fusion (ie, a receptor domain fused to an antibody Fc region, eg, as a dimer), wherein the receptor specifically binds to human PD-1.

-   18. The ligand of aspect 17, wherein the ligand is fully human. -   19. The ligand of aspect 17 or 18, wherein the ligand is for     administration by injection or the ligand is provided by an     injectable preparation; wherein the ligand is for topical or inhaled     administration or the ligand is provided by a topical or inhalable     preparation.

Optionally in any of the configurations, concepts, aspects, embodiments or examples, one or more of the following optional features applies.

Optionally, the cancer is selected from the group consisting of renal cell carcinoma; haematological neoplasm; stage IV melanoma; non small-cell lung cancer; metastatic stomach cancer; breast cancer; solid tumour; bladder cancer; head and neck cancer, colorectal cancer, glioblastoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, cervical cancer, myeloid leukaemia (eg, acute myeloid leukaemia or chronic myeloid leukaemia). In an embodiment, the cancer is not melanoma. In an embodiment, the cancer is not lung cancer. In an embodiment, the cancer is not non small-cell lung cancer.

Optionally, the cancer is renal cell cancer.

Optionally, the cancer is locally advanced or metastatic carcinoma, locally advanced or metastatic melanoma, or locally advanced or metastatic non-small cell lung carcinoma.

Optionally, the cancer is urothelial cancer.

Optionally, the cancer is recurrent or metastatic head and neck squamous cell carcinoma.

Optionally, the cancer is PD-L1-positive advanced or metastatic non-small cell lung cancer.

Optionally, the cancer is multiple myeloma.

Optionally, the cancer is advanced melanoma.

Optionally, the cancer is advanced Merkel cell cancer.

Optionally, the cancer is solid tumour, eg, advanced solid tumours.

In an example, the ligand comprises a human IgG4PE constant region.

In an example, the ligand is pembrolizumab, lambrolizumab or KEYTRUDA™. In an example, the ligand is nivolumab or OPDIVO™.

The TOI polymorphism is associated, for example, with a cancer, such as any cancer disclosed herein.

The invention also provides an anti-TOI ligand (eg, a human PD-1, PD-L1 or PD-L2 trap, or anti-human PD-1, PD-L1 or PD-L2 antibody or fragment) for use in any cancer treatment or immunotherapy method of the invention.

Optionally, the ligand (eg, an antibody or antibody fragment) is according to one of the following options:—

(i) wherein the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23*04), a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40. (ii) wherein the ligand comprises a VH domain derived from the recombination of human VH segment selected from the group consisting of IGHV3-33*01 and IGHV3-7*01, a human D gene segment and a human JH segment, and wherein said human comprises said selected VH gene segment or the human expresses VH domains derived from the recombination of said selected human VH segment, a human D gene segment and a human JH segment. (iii) wherein the ligand comprises a Vκ domain derived from the recombination of human Vκ segment selected from the group consisting of IGKV3-11*01 and IGKV1-12*01 and a human Jκ segment, and wherein said human comprises said selected Vκ gene segment or the human expresses Vκ domains derived from the recombination of said selected human Vκ segment and a human Jκ segment. (iv) wherein the ligand comprises a Vκ domain derived from the recombination of a human Vκ segment and a human Jκ segment, the human Vκ segment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36 and wherein said human comprises a Vκ gene segment encoding a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the human expresses Vκ domains that comprise a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 and wherein said human comprises a Vκ gene segment encoding a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 or the human expresses Vκ domains that comprise a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38.

Optionally, instead of a gamma-4 constant region, the ligand (eg, a trap, antibody or fragment) comprises a heavy chain constant region according to any of the following options:—

(v) wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4. (vi) wherein the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6. In an example, the constant region is Fe enhanced, eg, ADCC or CDC enhanced. The skilled person will know techniques (eg, known C region mutations) to enhance ADCC or CDC. (vii) wherein the ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3*01) constant region gene segment and wherein said human comprises (i) said first constant region gene segment (eg, an IGHG3*01), or the human expresses antibodies comprising human gamma-3 heavy chain constant regions encoded by said first human IGHG3 (eg, IGHG3*01) constant region gene segment.

Optionally, the ligand (eg, an antibody or antibody fragment) is according to one of the following options:—

(viii) wherein the ligand comprises a human kappa chain constant region that comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16 and wherein said human comprises (i) an IGKC1*01 human kappa chain constant region gene segment, or the human expresses antibodies comprising human kappa chain constant regions comprising a Val corresponding to position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16. (ix) wherein the ligand comprises a human IGLC1*01 lambda chain constant region and wherein said human comprises (i) a human IGLC1*01 lambda chain constant region gene segment, or the human expresses antibodies comprising human IGLC1*01 lambda chain constant regions. (x) wherein the ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human epsilon heavy chain constant regions encoded by said first constant region gene segment. (xii) wherein the ligand comprises a human mu heavy chain constant region encoded by a first human mu heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human mu heavy chain constant regions encoded by said first constant region gene segment. (xiii) wherein the ligand comprises a human alpha heavy chain constant region encoded by a first human alpha heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human alpha heavy chain constant regions encoded by said first constant region gene segment. (xiv) wherein the ligand comprises a human delta heavy chain constant region encoded by a first human delta heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human delta heavy chain constant regions encoded by said first constant region gene segment. (xv) wherein the ligand comprises a human kappa light chain constant region encoded by a first human kappa light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human kappa light chain constant regions encoded by said first constant region gene segment. (xvi) wherein the ligand comprises a human lambda light chain constant region encoded by a first human lambda light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human lambda light chain constant regions encoded by said first constant region gene segment.

REFERENCES

-   (a) Cancer Immunol Res. 2014 September; 2(9):846-56. doi:     10.1158/2326-6066.CIR-14-0040. Epub 2014 May 28; “In vitro     characterization of the anti-PD-1 antibody nivolumab, BMS-936558,     and in vivo toxicology in non-human primates”, Wang C et al. -   (b) Clin Biochem. 2014 May; 47(7-8):612-7. doi:     10.1016/j.clinbiochem.2013.12.023. Epub 2014 Jan. 2; “Programmed     death-1 (PD-1) polymorphisms in Chinese patients with esophageal     cancer”; Qiu H et al. -   (c) Gene. 2012 Oct. 25; 508(2):229-32. doi:     10.1016/j.gene.2012.07.059. Epub 2012 Aug. 8; “Programmed death-1     gene polymorphism (PD-1.5 C/T) is associated with colon cancer”;     Mojtahedi Z et al. -   (d) Gastroenterol Hepatol Bed Bench. 2013 Fall; 6(4):178-82;     “Programmed death-1 gene polymorphism (PD-1.5 C/T) is associated     with gastric cancer”; Savabkar S et al. -   (e) Breast Cancer Res Treat. 2011 August; 129(1):195-201. doi:     10.1007/s10549-011-1440-3. Epub 2011 Apr. 13; “PD-1 polymorphisms     are associated with sporadic breast cancer in Chinese Han population     of Northeast China”; Hua Z et al.

Human Polymorphisms & SNPs

The inventor carried out assessment of natural human variation in PD-1 and identified the following variations of interest for application to the present invention.

TABLE 23 Human PD-1 Variation CHROMOSOMAL LOCATION AMINO AMINO (FORWARD NUCLEOTIDE ACID ACID CUMULATIVE SNP/VARIANT STRAND) CHANGE¹ CHANGE² POSITION³ FREQUENCY⁴ rs7568402 rs28699177 rs28542728 rs118117097 rs28570544 rs118027315 rs191919871 rs6707556 rs142909968 rs36084323⁺ 2: 241859444 C > T 0.17 rs10204225 2: 197610964 G > A rs11568821⁺⁺ 2: 241851760 C > T 0.05 rs2227981⁺⁺⁺ 2: 241851121 G > A 0.38 rs2227982⁺⁺⁺⁺ 2: 241851281 G > A A215V 215 0.160 ^(1,2,3,4)According to Ensembl, dbSNP; with reference to transcript ENST00000334409 (the sequence of this transcript being incorporated herein in its entirety). ⁺rs36084323 is present in humans at an average cumulative frequency of 17% according to the 1000 Genomes Phase I database, but is higher in AMR, ASN, MXL, CHB, CHS and JPT populations; thus in an embodiment, the human is of ASN ancestry, eg, of MXL, CHB, CHS or JPT ancestry. ⁺⁺rs11568821 is present in humans at an average cumulative frequency of 5% according to the 1000 Genomes Phase I database, but is higher in AMR, EUR, PUR, CEU, GBR, IBS and TSI populations; thus in an embodiment, the human is of AMR or EUR ancestry, eg, of CEU, GBR, IBS or TSI ancestry. ⁺⁺⁺rs2227981 is present in humans at an average cumulative frequency of 38% according to the 1000 Genomes Phase I database, but is higher in AFR, EUR, ASW, LWK, YRI, CEU, GBR, IBS and TSI populations; thus in an embodiment, the human is of AFR or EUR ancestry, eg, of ASW, LWK, YRI, CEU, GBR, IBS or TSI ancestry. ⁺⁺⁺rs2227982 is present in humans at an average cumulative frequency of 16% according to the 1000 Genomes Phase I database, but is higher in ASN, CHB, CHS and JPT populations; thus in an embodiment, the human is of ASN ancestry, eg, of CHB, CHS or JPT ancestry.

Using this analysis, the inventor has devised the configurations, aspects, concepts, examples and embodiments of the invention set out in this Example 7 that are useful for addressing diseases and conditions, such as cancer, autoimmune or inflammatory diseases and conditions.

In an embodiment, the human comprises a SNP selected from the SNPs in column 1 of Table 23; optionally also the ligand specifically binds a PD-1 encoded by a nucleotide sequence comprising said SNP.

In an embodiment, the ligand (eg, trap, antibody or antibody fragment) specifically binds a PD-1 comprising a corresponding variation as shown in Table 23 or is encoded by a nucleotide sequence comprising or encoding a SNP or amino acid variation shown in Table 23.

For example the SNP is selected from rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982. For example the SNP is rs36084323. For example the SNP is rs10204225. For example the SNP is rs11568821. For example the SNP is rs2227981. For example the SNP is rs2227982.

In an example the SNP and indication are a SNP/indication combination mentioned in any of the references disclosed in this Example 7.

In an example, the indication is aplastic anaemia and the SNP is a G at the position of rs36084323.

Example 7B: Autoimmune, Inflammatory & Other Disease Applications

In an alternative, instead of “A method of cancer immunotherapy” or “A method of treating cancer”, the invention as expressed in Example 7A can instead be read as “A method of treating or reducing the risk of an autoimmune disease or condition” or “A method of treating or reducing the risk inflammatory disease or condition” and the TOI polymorphism is associated with an autoimmune or inflammatory disease or condition. The other non-cancer features of this example apply mutatis mutandis to these alternatives. The TOI (eg, PD-1) comprises said first polymorphism or is encoded by a nucleotide sequence comprising said selected SNP.

For example, the autoimmune disease or condition is selected from the group consisting of rheumatoid arthritis, diabetes (eg, type I diabetes mellitus), Grave's disease, multiple sclerosis and systemic lupus erythematosus.

For example, the inflammatory disease or condition is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, psoriasis, asthma, colitis and atopic dermatitis. For example, the method is for treating or reducing the risk of ankylosing spondylitis. In an example, the human is of Chinese (eg, CHS) ancestry.

REFERENCES

-   (a) Vibanco-Pérez N, Gutiérrez-Franco J, Duran-Avelar M,     Agraz-Cibrian J, Jiménez-Alvarez M, Pena-Virgen S and     Zambrano-Zaragoza J (2013). Association of PD1.1 and PD1.6     polymorphisms with ankylosing spondylitis. Front. Immunol.     Conference Abstract: 15th International Congress of Immunology     (ICI). doi: 10.3389/conf.fimmu.2013.02.0079. Various SNPs in the PD1     gene have been identified, such as PD1.1 (rs36084323), PD1.3     (rs11568821), PD1.5 (rs2227981), and PD1.9 (rs2227982). Among these,     PD1.3, PD1.5, and PD1.9 have been associated with autoimmune     disorders in different human populations. PD1.1 and PD1.6 are two     single nucleotide polymorphisms, at −600 G/A (rs36084323), and +8669     G/A (rs10204225) respectively, that have been associated with others     autoimmune diseases such as rheumatoid arthritis, type I diabetes     mellitus, and systemic lupus erythematosus. -   (b) Rheumatol Int. 2011 February; 31(2):209-13. doi:     10.1007/s00296-009-1264-1. Epub 2009 Dec. 12; “Programmed cell death     1 gene polymorphisms is associated with ankylosing spondylitis in     Chinese Han population”; Liu X et al.

The genotype distributions of PD-1.9 were reportedly significantly different between the patients with ankylosing spondylitis and the controls (P=0.025). The frequencies of TC genotype and T allele of PD-1.9 were higher in the patients than those in the controls (P=0.026 and 0.004). Moreover, the frequency of the CT haplotype (PD-1.5 C/T, PD-1.9 T/C) was significantly higher in AS patients than the controls (21.6 vs. 13.9%, P=0.002). The CC haplotype was more common in the controls than in the patients.

-   (c) Tissue Antigens. 2003 December; 62(6):492-7; “Association of a     putative regulatory polymorphism in the PD-1 gene with     susceptibility to type 1 diabetes”, Nielsen C et al. Human PD-1     intronic 7146G/A SNP was found to be associated with the development     of type 1 diabetes in humans. -   (d) Inflammation. 2011 December; 34(6):707-12. doi:     10.1007/s10753-010-9282-4; “Study of programmed cell death 1 (PDCD1)     gene polymorphims in Iranian patients with ankylosing spondylitis”,     Soleimanifar N et al. -   (e) Clin Exp Rheumatol. 2011 January-February; 29(1):13-8. Epub 2011     Feb. 23; “Association of polymorphisms in the programmed cell death     1 (PD-1) and PD-1 ligand genes with ankylosing spondylitis in a     Chinese population”; Yang Q et al. -   (f) Arthritis Rheum. 2005 June; 52(6):1665-9; “Association of a     PDCD1 polymorphism with renal manifestations in systemic lupus     erythematosus”; Johansson M et al. -   (g) Arthritis Rheum. 2005 April; 52(4):1058-62; “A new haplotype of     PDCD1 is associated with rheumatoid arthritis in Hong Kong Chinese”;     Kong E K et al. -   (h) Ann Neurol. 2005 July; 58(1):50-7; “A PD-1 polymorphism is     associated with disease progression in multiple sclerosis”; Kroner A     et al. -   (i) Leuk Lymphoma. 2013 October; 54(10):2251-4. doi:     10.3109/10428194.2013.772605. Epub 2013 Mar. 4; “Association between     polymorphisms in PDCD1 gene and aplastic anemia in Chinese Han     population; Wu Z et al.

Example 8: Iron Regulation

The existence of multiple inherited disorders of iron metabolism suggests genetic contributions to iron deficiency. To this end, the inventor assessed naturally-occurring variation in human proteins of the iron homeostasis pathway, and for example the hemojuvelin-BMP6-hepcidin-ferroportin axis.

BMP6 is also known as bone morphogenetic protein 6, BMP-6, VGR-1, VG-1-R, VG-1-related protein, Vg1-related sequence, vegetal-related (TGFB related) cytokine and vegetal related growth factor (TGFB-related).

Matriptase-2 (also known as TMPRSS6) is a critical regulator of the iron-regulatory hormone hepcidin in the liver; matriptase-2 cleaves membrane-bound hemojuvelin and consequently alters bone morphogenetic protein (BMP) signaling. Hemojuvelin and hepcidin play a critical role in retinal iron homeostasis; they are also expressed in the retina.

The invention provides methods of iron homeostasis involving administering anti-TOI ligand to a human, the TOI being present in humans as a plurality of variants differing by one or more amino acid polymorphisms, the method comprising administering a ligand to the human, the ligand comprising first and second protein domains, wherein the first domain specifically binds a TOI variant comprising a first amino acid polymorphism, wherein the second domain comprises a second polymorphism, and wherein the human expresses (i) TOI comprising said first amino acid polymorphism; and (ii) protein domains comprising said second polymorphism. Particularly useful are methods wherein the TOI is selected from the human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis.

A: Indications & Humans

Optionally the method is a method for increasing or maintaining increased blood iron, eg, for treating or reducing the risk of anaemia; or treating iron deficiency; or for treating or reducing the risk of Anaemia of Chronic Inflammation (ACI); or for treating or reducing the risk of Anaemia of Chronic Disease (ACD); or for treating or reducing the risk of anaemia associated with cancer, a kidney condition or GvHD; or for treating or reducing the risk of hemochromatosis.

In an embodiment, the patient is a haemodialysis patient and optionally TOI is selected from the human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis (eg, the TOI is TMPRSS6 that comprises the A736V polymorphism; or eg, the human comprises the A736V polymorphism).

In an embodiment, the patient is a coronary heart disease patient. In an embodiment, the human is suffering from an inflammatory disease or condition, eg, an acute acute inflammation.

In an embodiment, the method is for treating or reducing the risk of haemochromatosis.

In an embodiment, the method is for reducing or maintaining reduced hepcidin level in the human.

In an embodiment, the method is for regulating (eg, increasing; eg, decreasing) erythropoiesis in the human and optionally TOI is selected from the human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis (eg, the TOI is TMPRSS6 that comprises the A736V polymorphism; or eg, the human comprises the A736V polymorphism).

In an embodiment, the method is for treating or reducing the risk of iron overload in the human.

In an embodiment, the method is for treating or reducing the risk of iron-refractory iron deficiency anemia (IRIDA) in the human.

B: Human Polymorphisms & SNPs

Any feature of this section B is combinable with any feature of section A above. For example, the human and/or TOI comprises a SNP as described below and the method or ligand of the invention is according to one or more features of A, comprising administering an anti-TOI ligand.

The inventor carried out assessment of natural human variation in genes and proteins involved in iron homeostasis in humans and identified the following variations of interest for application to the present invention.

TABLE 24 Iron Homeostasis - Human Polymorphism & SNPs CHROMOSOMAL LOCATION AMINO (FORWARD NUCLEOTIDE ACID AMINO ACID CUMULATIVE SNP/VARIANT STRAND) CHANGE¹ CHANGE² POSITION³ FREQUENCY⁴ BMP6⁵ rs111588693*  6: 7727038 G > A R28Q 28 0.338 TRANSFERRIN rs3811647**  3: 133765185 G > A 0.34 TMPRS6⁶ Y141C I212T R271Q S304L C510S rs855791*** 22: 37066896 A > G A736V 736 0.398 rs1421312**** 22: 37091770 A > G rs2543519 22: 37084229 A > G I430T 0.245 rs2235324 22: 37089684 T > C K244W 244 0.393 HFE⁷ rs1799945  6: 26090951 C > G H63D 63 0.084 rs1800562  6: 26092913 G > A C282Y 282 0.020 CUBN⁸ rs10904850# 10: 16955708 G > A 0.21 rs1801224 10: 17105522 T > G P389T 389 0.440 FERROPORTIN⁹ rs11568350##  2: 189565370 C > A Q248H 248 0.010 HAEMQJUVELIN¹⁰ rs7540883###  1: 146018429 C > G A310G 310 0.022 HEPCIDIN¹¹ rs146776859 19: 35284790 C > T T31M 31 0.002 MISCELLANEOUS rs987710 22: 22158022 G > A 0.38 rs2698530  2: 64276761 A > C 0.33 ^(1,2,3,4)According to Ensembl, dbSNP; ⁵with reference to transcript ENST00000283147 (the sequence of this transcript being incorporated herein in its entirety). ⁶with reference to transcript ENST00000346753 (the sequence of this transcript being incorporated herein in its entirety); with the exception of rs2543519, which is with reference to transcript ENST00000442782 (the sequence of this transcript being incorporated herein in its entirety); ⁷with reference to transcript ENST00000309234 (the sequence of this transcript being incorporated herein in its entirety); ⁸with reference to transcript ENST00000377833 (the sequence of this transcript being incorporated herein in its entirety); ⁹with reference to transcript ENST00000261024 (the sequence of this transcript being incorporated herein in its entirety); ¹⁰with reference to transcript ENST00000336751 (the sequence of this transcript being incorporated herein in its entirety); ¹¹with reference to transcript ENST00000222304 (the sequence of this transcript being incorporated herein in its entirety); *rs111588693 is present in humans at an average cumulative frequency of 34% according to the 1000 Genomes Phase I database, but is higher in EUR, CEU, FIN, GBR, IBS and TSI populations; thus in an embodiment, wherein the method involves BMP6, eg administering an anti-BMP6 ligand, the human is of EUR ancestry, eg, of CEU, FIN, GBR, IBS or TSI ancestry. **rs3811647 is present in humans at an average cumulative frequency of 34% according to the 1000 Genomes Phase I database, but is higher in ASN, CHB, CHS and JPT populations; thus in an embodiment, wherein the method involves transferrin, eg administering an anti-transferrin ligand, the human is of ASN ancestry, eg, of CHB, CHS or JPT ancestry. ***rs855791 is present in humans at an average cumulative frequency of 40% according to the 1000 Genomes Phase I database, but is higher in AMR, ASN, CLM, MXL, PUR, CHB, CHS and JPT populations; thus in an embodiment, wherein the method involves TMPRSS6, eg administering an anti-TMPRSS6 ligand, the human is of AMR or ASN ancestry, eg, of CLM, MXL, PUR, CHB, CHS or JPT ancestry. ****rs1421312 is present in humans at an average cumulative frequency of 44% according to the 1000 Genomes Phase I database, but is higher in AFR, ASW, LWK and YRI populations; thus in an embodiment, wherein the method involves TMPRSS6, eg administering an anti-TMPRSS6 ligand, the human is of AFR ancestry, eg, of ASW, LWK or YRI ancestry. #rs10904850 is present in humans at an average cumulative frequency of 21% according to the 1000 Genomes Phase I database, but is higher in EUR, CEU, FIN, GBR, IBS and TSI populations; thus in an embodiment, wherein the method involves CUBN, eg administering an anti-CUBN ligand, the human is of EUR ancestry, eg, of CEU, FIN, GBR, IBS or TSI ancestry. ##rs11568350 is present in humans at an average cumulative frequency of 1% according to the 1000 Genomes Phase I database, but is higher in AFR, ASW, LWK and YRI populations; thus in an embodiment, wherein the method involves ferroportin, eg administering an anti-ferroportin ligand (eg, antibody), the human is of AFR ancestry, eg, of, ASW, LWK or YRI ancestry. ###rs7540883 is present in humans at an average cumulative frequency of 2% according to the 1000 Genomes Phase I database, but is higher in AFR, ASW, LWK and YRI populations; thus in an embodiment, wherein the method involves haemojuvelin, eg administering an anti-haemojuvelin ligand (eg, antibody or trap, such as human HJV-Fc dimer), the human is of AFR ancestry, eg, of, ASW, LWK or YRI ancestry.

SNP rs111588693 encodes an amino acid in the region of the cleavage site for the proprotein of BM6, and thus it may be desirable to target a BMP6 encoded by a nucleotide sequence comprising rs111588693 in the human, optionally wherein the human comprises a BMP6 nucleotide sequence comprising said SNP.

In an embodiment, the human comprises a SNP selected from the SNPs in column 1 of Table 24; optionally also the ligand specifically binds a protein (eg, a TOI of said human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis) encoded by a nucleotide sequence comprising said SNP.

In an embodiment, the ligand (eg, trap, antibody or antibody fragment) specifically binds a TOI protein (eg, a TOI of said human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis) comprising a corresponding variation as shown in Table 24 or is encoded by a nucleotide sequence comprising or encoding a SNP or amino acid variation shown in Table 24.

For example, the TOI is a protein of the human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis. In an example, the TOI is human hemojuvelin. In an example, the TOI is human BMP6 (and optionally comprising amino acid 28Q or encoded by a BMP6 nucleotide sequence comprising SNP rs111588693; in an embodiment the human expresses BMP6 that comprises said variation). In an example, the TOI is human hepcidin. In an example, the TOI is human ferroportin. In an example, the TOI is human transferrin (and optionally encoded by a transferrin nucleotide sequence comprising SNP rs3811647; in an embodiment the human expresses transferrin that comprises said variation). In an example, the TOI is human TMPRSS6 (and optionally comprises one or more variations selected from the group consisting of A736V, Y141C, 1212T, R271Q, S304L and C510S; or is encoded by a TMPRSS6 nucleotide sequence comprising SNP rs855791 or rs1421312 or encoding one or more variations selected from said group; in an embodiment the human expresses TMPRSS6 that comprises said variation). For example, the TMPRSS6 comprises variation A736V and optionally the human expresses TMPRSS6 that comprises variation A736V. In an example, the TOI is human erythroferrone. In an example, the TOI is human hemochromatosis protein, HFE (and optionally comprising amino acid 282Y or encoded by a HFE nucleotide sequence comprising SNP rs1800562; in an embodiment the human expresses HFE that comprises said variation). In an example, the TOI is the product of the human CUBN nucleotide sequence or the human comprises a CUBN nucleotide sequence, the nucleotide sequence comprising SNP rs10904850; in an embodiment the human expresses CUBN protein that comprises said variation. CUBN is also known as cubilin and intrinsic factor-cobalamin receptor. Ferroportin is also known as solute carrier family 40 member 1 (SLC40A1) or iron-regulated transporter 1. Haemojuvelin is also known as hemojuvelin, HFE2A, HJV, JH and RGMC.

In an example, the human comprises SNP rs2698530 on chr. 2p14 and/or rs987710 on chr. 22q11.

It may be desirable to block (not necessarily clear as with an IgG1 format) the TOI in the method of this alternative and a human IgG4 may be preferable for doing this in an iron regulation setting. Thus, in an embodiment, the ligand comprises a human gamma-4 constant region. For the iron regulation setting of the invention, it may be advantageous wherein the gamma-4 is a gamma-4 constant region with 228Pro or 235Glu. In an example the constant region is an IgG4PE (ie, a gamma-4 constant region with 228Pro and 235Glu). In an example, the ligand comprises human IGHG4*01 hinge S10>P constant region.

In an example, therefore, the ligand comprises any human gamma-4 constant region disclosed herein (eg, in any of Examples 1 et seq).

REFERENCES

-   (a) Nutr Hosp. 2012 November-December; 27(6):2142-5. doi:     10.3305/nh.2012.27.6.6154; “Intronic SNP rs3811647 of the human     transferrin gene modulates its expression in hepatoma cells”;     Blanco-Rojo R et al.

Transferrin (Tf) exerts a crucial function in the maintenance of systemic iron homeostasis. The expression of the Tf gene is controlled by transcriptional mechanism. The authors conclude that A allele of SNP rs3811647 increases Tf expression in a manner that might underlie inter-individual variation in serum transferrin levels observed in different population groups.

-   (b) J Med Genet. 2013 September; 50(9):593-8. doi:     10.1136/jmedgenet-2013-101673. Epub 2013 Jun. 21; “Associations of     common variants in HFE and TMPRSS6 with iron parameters are     independent of serum hepcidin in a general population: a replication     study”; Galesloot T E et al.

The authors found that HFE rs1800562 and TMPRSS6 rs855791 are the main determinants of HFE and TMPRSS6 related variation in serum iron, ferritin, transferrin saturation, and total iron binding capacity.

-   (c) BMC Nephrol. 2013 Feb. 22; 14:48. doi: 10.1186/1471-2369-14-48;     “The A736V TMPRSS6 polymorphism influences hepcidin and iron     metabolism in chronic hemodialysis patients: TMPRSS6 and hepcidin in     hemodialysis”; Pelusi S et al.

The A736V TMPRSS6 polymorphism is a major genetic determinant of iron metabolism in healthy subjects. The authors reported the TMPRSS6 736 V variant was associated with higher hepcidin levels (p=0.017). In patients without acute inflammation and overt iron deficiency (C reactive protein <1 mg/dl and ferritin >30 ng/ml; n=86), hepcidin was associated with lower mean corpuscular volume (p=0.002), suggesting that it contributed to iron-restricted erythropoiesis. In line with previous results, in patients without acute inflammation and severe iron deficiency the “high hepcidin” 736 V TMPRSS6 variant was associated with higher erythropoietin maintenance dose (p=0.016), independently of subclinical inflammation (p=0.02).

-   (d) PLoS One. 2012; 7(6):e38339. doi: 10.1371/journal.pone.0038339.     Epub 2012 Jun. 22; “Associations between single nucleotide     polymorphisms in iron-related genes and iron status in multiethnic     populations”; McLaren C E et al.

Three chromosomal regions showed association across multiple populations, including SNPs in the TF and TMPRSS6 genes, and on chromosome 18q21. A novel SNP rs1421312 in TMPRSS6 was associated with serum iron in whites (p=3.7×10(−6)) and replicated in African Americans (p=0.0012). Twenty SNPs in the TF gene region were associated with total iron-binding capacity in whites (p<4.4×10(−5)); six SNPs replicated in other ethnicities (p<0.01). SNP rs10904850 in the CUBN gene on 10p13 was associated with serum iron in African Americans (P=1.0×10(−5)).

-   (e) PLoS One. 2011 Mar. 31; 6(3):e17390. doi:     10.1371/journal.pone.0017390; “Genome-wide association study     identifies genetic loci associated with iron deficiency”; McLaren C     E et al. Five SNPs were identified in a Genome-Wide Association     Study that met genome-wide statistical significance for association     with at least one iron measure, rs2698530 on chr. 2p14; rs3811647 on     chr. 3q22, a known SNP in the transferrin (TF) gene region;     rs1800562 on chr. 6p22, the C282Y mutation in the HFE gene;     rs7787204 on chr. 7p21; and rs987710 on chr. 22q11 (GWAS observed     P<1.51×10(−7) for all). An association was observed between total     iron binding capacity and SNP rs3811647 in the TF gene (GWAS     observed P=7.0x 10(−9). The analysis revealed strong associations     between rs2698530 on chr. 2p14 and iron status outcomes. -   (f) Hum Mutat. 2010 May; 31(5):E1390-405. doi: 10.1002/humu.21243;     “Novel TMPRSS6 mutations associated with iron-refractory iron     deficiency anemia (IRIDA)”; De Falco L et al. Mutations leading to     abrogation of matriptase-2 proteolytic activity in humans are     associated with an iron-refractory iron deficiency anemia (IRIDA)     due to elevated hepcidin levels. The authors describe 12 IRIDA     patients belonging to 7 unrelated families and identify 10 (9 novel)     TMPRSS6 mutations spread along the gene sequence: 5 missense, 1 non     sense and 4 frameshift. The frameshift and non sense mutations are     predict to result in truncated protein lacking the catalytic domain.     The causal role of missense mutations (Y141C, I212T, R271Q, S304L     and C510S) is demonstrated by in silico analysis, their absence in     100 control chromosomes and the high conservation of the involved     residues. The C510S mutation in the LDLRA domain in silico model     causes an intra-molecular structural imbalance that impairs     matriptase-2 activation. We also assessed the in vitro effect on     hepcidin promoter and the proteolytic activity of 1212T and R271Q     variants demonstrating a reduced inhibitory effect for the former     mutation, but surprisingly a normal function for R271Q which appears     a silent mutation in vitro. Based on mRNA expression studies 1212T     could also decrease the total amount of protein produced, likely     interfering with mRNA stability. Collectively, our results extend     the pattern of TMPRSS6 mutations associated with IRIDA and propose a     model of causality for some of the novel missense mutation.

The invention provides the following concepts:—

-   1. A method of treating or reducing the risk of a disease or     condition in a human, wherein the disease or condition is mediated     by a TOI, the TOI being present in humans as a plurality of variants     differing by one or more amino acid polymorphisms, the method     comprising administering a ligand (eg, an antibody or antibody     fragment) to the human, the ligand comprising first and second     protein domains, wherein the first domain specifically binds a TOI     variant comprising a first amino acid polymorphism, wherein the     second domain comprises a second polymorphism, and wherein the human     expresses (i) TOI comprising said first amino acid polymorphism;     and (ii) protein domains comprising said second polymorphism,     optionally wherein the ligand comprises a human gamma-4 heavy chain     constant region that comprises a Leu at position 189 shown in SEQ ID     NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein     said human comprises an IGHG4*01 human heavy chain constant region     gene segment, or the human expresses antibodies comprising human     gamma-4 heavy chain constant regions comprising a Leu at position     189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID     NO: 73; and wherein the second domain is comprised by said gamma-4     heavy chain constant region of the ligand.

Optionally, the TOI is selected from the human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis. In an example, the TOI is human hemojuvelin. In an example, the TOI is human BMP6. In an example, the TOI is human TMPRSS6. In an example, the TOI is human hepcidin. In an example, the TOI is human ferroportin. In an example, the TOI is human HFE. In an example, the TOI is human transferrin. A TOI of “the human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis” is a human protein (eg, expressed by liver) that is part of the iron regulation pathway comprising hemojuvelin, BMP6, TMPRSS6, hepcidin, ferroportin and transferrin.

For example, the method can be for any of the purposes or indications set out section A. In an example, the method is for regulating iron in the human. In an example, the iron is blood iron or circulating iron or free iron.

In an example, the method is a method of reducing anaemia in the human.

-   2. The method of concept 1, wherein the first and/or second     polymorphism is respectively encoded by a SNP having a cumulative     human allele frequency of less than 50%.

Optionally the frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

-   3. The method of any preceding concept, wherein the first domain is     an antibody variable domain or a receptor domain. -   4. The method of any preceding concept, wherein the second domain is     an antibody domain, optionally an antibody variable or constant     domain. -   5. The method of any preceding concept, wherein the second domain is     a domain of an antibody Fc region. -   6. The method of any one of concepts 1 to 4, wherein the first and     second domains are antibody variable domains, optionally wherein the     variable domains specifically bind first and second human TOI     respectively, wherein the TOIs are different.

Optionally, the second TOI is selected from the human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis. In an example, the second TOI is human hemojuvelin. In an example, the second TOI is human BMP6. In an example, the second TOI is human TMPRSS6. In an example, the second TOI is human hepcidin. In an example, the second TOI is human ferroportin. In an example, the second TOI is human HFE. In an example, the second TOI is human transferrin.

-   7. The method of any one of concepts 1 to 4, wherein the first and     second domains are receptor domains, optionally wherein the receptor     domains specifically bind first and second human TOI respectively,     wherein the TOIs are different. -   8. The method of concept 6 or 7, wherein the first domain comprises     a third amino acid polymorphism, wherein the human expresses (iii)     domains comprising said third amino acid polymorphism.

Optionally the human expresses the second TOI variant.

Optionally the third polymorphism is encoded by a SNP having a cumulative human frequency of less than 50%. Optionally the frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

In an example, the first domain is an antibody variable domain (eg, any V domain of the invention described herein, such human VH3-23*04 as herein described) and the domains of (iii) are antibody variable domains. For example, the first domains and domains of (iii) are VH domains. For example, the first domains and domains of (iii) are Vκ domains. For example, the first domains and domains of (iii) are Vλ domains.

In an example, the first domain is a receptor binding site and the domains of (iii) are said receptor binding site domains.

-   9. A method according to concepts 1, 2 and 8 combined. -   10. The method of any preceding concept, wherein the human expresses     the first and/or second domain. -   11. The method of any preceding concept, wherein the first, second     and/or third polymorphism is respectively found in at least 5     different human populations as defined in Table 4.

For example, each of the first and second polymorphisms is respectively found in at least 5 different human populations as defined in Table 4. For example, each of the first, second and polymorphisms is respectively found in at least 5 different human populations as defined in Table 4.

Optionally the first, second and/or third (see below) polymorphism respectively appears in at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different human populations as defined in Table 4.

-   12. The method of concept 11, wherein the first, second and/or third     polymorphism respectively appears in at least 15 humans in the 1000     Genomes Phase I database.

In an embodiment, the first, second and/or third polymorphism is found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 human individuals distributed across such many different human populations.

-   13. The method of any preceding concept, wherein the first, second     and/or third polymorphism is respectively found in at least 5     different human populations as defined in Table 4 and is encoded by     a SNP having a cumulative human allele frequency of less than 50%.

Optionally the frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

-   14. The method of any preceding concept, wherein the first, second     and/or third polymorphism is respectively encoded by a SNP having     total human genotype frequency of less than 50%.

Optionally the frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

-   15. The method of any preceding concept, wherein the first and     second domains are human, optionally wherein the ligand is fully     human. -   16. The method of any preceding concept, wherein the ligand is an     anti-TOI trap, antibody or antibody fragment. -   17. The method of any preceding concept, wherein the ligand     specifically binds two or more human TOIs, eg, said first and second     TOIs. -   18. The method of any preceding concept, wherein the ligand is     administered to the human by injection or the ligand is provided by     an injectable preparation. -   19. The method of any preceding concept, wherein said human has been     or is genotyped as positive for said TOI variant nucleotide sequence     before administering the ligand, or the method comprises genotyping     the human as positive for said variant nucleotide sequence before     administering the ligand. -   20. The method of any preceding concept, wherein the human has been     or is phenotyped as positive for said TOI variant before     administering the ligand, or the method comprises phenotyping the     human as positive for said variant before administering the ligand. -   21. The method of any preceding concept, comprising, before said     administering, selecting a human comprising said first TOI     polymorphism, wherein the human is the human of concept 1. -   22. The method of any preceding concept, wherein the human has been     determined to comprise the first and/or second TOI variant or a     nucleotide sequence encoding the TOI variant(s). -   23. The method of any preceding concept, comprising the step of     determining that the human comprises a first TOI nucleotide sequence     encoding the first polymorphism, optionally, wherein the determining     step is performed before administration of the ligand to the human. -   24. The method of concept 23 wherein the step of determining     comprises assaying a biological sample from the human for said     nucleotide sequence. -   25. The method of concept 24, wherein the assaying comprises     contacting the biological sample with     -   h. a. at least one oligonucleotide probe comprising a sequence         of at least 10 contiguous nucleotides that can specifically         hybridize to and identify in the biological sample a nucleotide         sequence comprising a SNP encoding the first polymorphism or         that specifically hybridizes to an antisense of said sequence,         wherein said nucleic acid hybridizes to said SNP or hybridizes         to an antisense sequence thereby forming a complex when at least         one nucleotide sequence comprising said SNP is present; and/or     -   i. b. at least one oligonucleotide probe comprising a sequence         of at least 10 contiguous nucleotides of a nucleotide sequence         comprising said SNP or comprising an antisense sequence of said         contiguous nucleotides, wherein said sequence of contiguous         nucleotides comprises said SNP thereby forming a complex when         the nucleotide sequence comprising said SNP is present; and     -   j. detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the first TOI nucleotide sequence. -   26. The method of concept 24, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   27. The method of any preceding concept, wherein said human is or     has been further determined to be substantially resistant to an     anaemia treatment. -   28. The method of any preceding concept, wherein said human is     receiving or has received an anaemia treatment or has reduced     responsiveness to an anaemia treatment. -   29. The method of concept 25, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   30. The method of any preceding concept, wherein said human has been     diagnosed with said disease or condition. -   31. An anti-TOI ligand (eg, an antibody, antibody fragment or trap)     for use in the method of any preceding concept.

In an embodiment, the trap is a receptor Fc fusion (ie, a receptor domain fused to an antibody Fc region, eg, as a dimer), wherein the receptor specifically binds to the TOI.

-   32. The ligand of concept 31, wherein the ligand is fully human. -   33. The ligand of concept 31 or 32, wherein the ligand is for     administration by injection or the ligand is provided by an     injectable preparation.

In an example, the first, second and/or third polymorphism respectively:—

-   -   k. has a cumulative human allele frequency of 15% or less;     -   l. has a total human genotype frequency of 20% or less;     -   m. is found in at least 5 different human populations (using the         standard categorisation of the 1000 Genomes Project, eg, as per         the Phase I database); and     -   n. is found in many individuals distributed across such many         different populations.

In an example, the criteria are applied with reference to one or more human genomic sequence databases as described herein. For example, the criteria are those as applied to the 1000 Genomes database.

For example in any aspect example, embodiment or configuration of the invention, the 1000 Genomes database release 13 or Phase I. For example, the 1000 Genomes database in its most recent version as at 1 Oct. 2013.

In an example, the human is homozygous for the first polymorphism. Additionally or alternatively, the human is homozygous for the first polymorphism. Additionally or alternatively, the human is homozygous for the third polymorphism. For example, the human is homozygous for the first and second polymorphisms.

In an example, the method is according to any one of the disclosure herein involving selecting, genotyping or phenotyping the human as positive for the TOI variant (ie, in the present case human PD-1 comprising the polymorphism or a nucleotide sequence encoding this). In an example, the method is according to any one of concepts above involving the determining step or wherein the human has been determined to comprise the variant TOI variant (eg, in the present case human PD-1 comprising the polymorphism or a nucleotide sequence encoding this).

In an example, the first polymorphism is a human BMP6 amino acid polymorphism described herein or is encoded by a human BMP6 SNP described herein.

In an example, wherein the TOI is human BMP6 the first polymorphism is encoded by SNP rs111588693.

The invention further provides the following specific aspects:—

-   -   1. A method of treating or reducing the risk of anaemia in a         human by targeting in the human a human TOI selected from the         group consisting of BMP6, ferroportin, haemojuvelin, hepcidin         and TMPRSS6, the method comprising administering an antibody or         antibody fragment to the human, wherein the antibody or antibody         fragment specifically binds a variant of said TOI comprising a         first amino acid polymorphism, wherein the antibody or antibody         fragment comprises a human gamma-4 heavy chain constant region         that comprises a Leu at position 189 shown in SEQ ID NO: 73 or         an Arg at position 289 shown in SEQ ID NO: 73 and wherein said         human comprises (i) an IGHG4*01 human heavy chain constant         region gene segment, or the human expresses antibodies         comprising human gamma-4 heavy chain constant regions comprising         a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at         position 289 shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide         sequence encoding a PD-1 comprising said first amino acid         polymorphism or the human expresses a PD-1 comprising said first         amino acid polymorphism.

Preferably, the trap, antibody or fragment of any configuration, concept, aspect, example or embodiment comprises an IgG4PE constant region.

In an example of any configuration, concept, aspect, example or embodiment, an anti-TOI (eg, anti-BMP6) antibody is used.

A first aspect provides:—

A method of treating or reducing the risk of anaemia in a human, the method comprising administering an anti-BMP6 trap, antibody or antibody fragment to the human, wherein the trap, antibody or antibody fragment specifically binds a human BMP6 encoded by a BMP6 nucleotide sequence comprising a SNP rs1 11588693, wherein the trap, antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a BMP6 nucleotide sequence comprising said SNP; optionally wherein the human has been determined to comprise said SNP or the method comprises determining that the human comprises said SNP before administering the antibody or fragment.

A second aspect provides:—

A method of treating or reducing the risk of anaemia in a human, the method comprising administering an anti-haemojuvelin trap, antibody or antibody fragment to the human, wherein the trap, antibody or antibody fragment specifically binds a human haemojuvelin encoded by a haemojuvelin nucleotide sequence comprising SNP rs7540883, wherein the trap, antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a haemojuvelin nucleotide sequence comprising said SNP; optionally wherein the human has been determined to comprise said SNP or the method comprises determining that the human comprises said SNP before administering the antibody or fragment.

A third aspect provides:—

A method of treating or reducing the risk of anaemia in a human, the method comprising administering an anti-ferroportin trap, antibody or antibody fragment to the human, wherein the trap, antibody or antibody fragment specifically binds a human ferroportin encoded by a ferroportin nucleotide sequence comprising SNP rs11568350, wherein the trap, antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a ferroportin nucleotide sequence comprising said SNP; optionally wherein the human has been determined to comprise said SNP or the method comprises determining that the human comprises said SNP before administering the antibody or fragment.

A fourth aspect provides:—

A method of treating or reducing the risk of anaemia in a human, the method comprising administering an anti-TMPRSS6 trap, antibody or antibody fragment to the human, wherein the trap, antibody or antibody fragment specifically binds a human TMPRSS6 encoded by a TMPRSS6 nucleotide sequence comprising a SNP selected from the group consisting of rs855791, rs1421312, rs2543519, rs2235324 wherein the trap, antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a TMPRSS6 nucleotide sequence comprising said selected SNP; optionally wherein the human has been determined to comprise said SNP or the method comprises determining that the human comprises said SNP before administering the antibody or fragment.

A fifth aspect provides:—

A method of treating or reducing the risk of anaemia in a human, the method comprising administering an anti-hepcidin trap, antibody or antibody fragment to the human, wherein the trap, antibody or antibody fragment specifically binds a human hepcidin encoded by a hepcidin nucleotide sequence comprising SNP rs146776859, wherein the trap, antibody or antibody fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii) a hepcidin nucleotide sequence comprising said SNP; optionally wherein the human has been determined to comprise said SNP or the method comprises determining that the human comprises said SNP before administering the antibody or fragment.

-   -   2. The method of aspect 1, wherein the human is homozygous for         said selected SNP.     -   3. The method of any preceding aspect, wherein the ligand         comprises an IGHG4*01 human heavy chain constant region, eg,         wherein the constant region is a gamma-4 constant region with         228Pro and/or 235Glu.     -   4. The method of any preceding aspect, wherein the anaemia is         selected from the group consisting of Anaemia of Chronic         Inflammation (ACI); Anaemia of Chronic Disease (ACD); or anaemia         associated with cancer, a kidney condition or GvHD.     -   5. The method of any preceding aspect, comprising, before said         administering, selecting a human comprising said BMP6,         ferroportin, haemojuvelin, TMPRSS6 or hepcidin nucleotide         sequence of (ii), wherein the human is the human of aspect 1.     -   6. The method of any preceding aspect, wherein the human has         been determined to comprise said BMP6, ferroportin,         haemojuvelin, TMPRSS6 or hepcidin nucleotide sequence of (ii).     -   7. The method of any preceding aspect, comprising the step of         determining that the human comprises said BMP6, ferroportin,         haemojuvelin, TMPRSS6 or hepcidin nucleotide sequence of (ii),         optionally, wherein the determining step is performed before         administration of the antibody to the human.     -   8. The method of aspect 7, wherein the step of determining         comprises assaying a biological sample from the human for         respectively a BMP6, ferroportin, haemojuvelin, TMPRSS6 or         hepcidin nucleotide sequence comprising said selected variation.     -   9. The method of aspect 8, wherein the assaying comprises         contacting the biological sample with         -   a. a. at least one oligonucleotide probe comprising a             sequence of at least 10 contiguous nucleotides that can             specifically hybridize to and identify in the biological             sample a nucleotide sequence comprising said selected             variation or that specifically hybridizes to an antisense of             said sequence, wherein said nucleic acid hybridizes to said             selected SNP or hybridizes to an antisense sequence thereby             forming a complex when at least one nucleotide sequence             comprising said selected SNP is present; and/or         -   b. b. at least one oligonucleotide probe comprising a             sequence of at least 10 contiguous nucleotides of a             nucleotide sequence comprising said selected SNP or             comprising an antisense sequence of said contiguous             nucleotides, wherein said sequence of contiguous nucleotides             comprises said selected SNP thereby forming a complex when             the nucleotide sequence comprising said selected SNP is             present; and         -   c. detecting the presence or absence of the complex, wherein             detecting the presence of the complex determines that the             human comprises respectively a BMP6, ferroportin,             haemojuvelin, TMPRSS6 or hepcidin nucleotide sequence             comprising said selected SNP.     -   10. The method of aspect 9, wherein the assaying comprises         nucleic acid amplification and optionally one or more methods         selected from sequencing, next generation sequencing, nucleic         acid hybridization, and allele-specific amplification and/or         wherein the assaying is performed in a multiplex format.     -   11. The method of any preceding aspect, wherein said human is or         has been further determined to be substantially resistant an         anaemia treatment (eg, EPO).     -   For example the human is or has been further determined to be         resistant to an anaemia treatment (eg, EPO).     -   12. The method of any preceding aspect, wherein said human is         receiving or has an anaemia treatment (eg, EPO) or has reduced         responsiveness to an anaemia treatment (eg, EPO).     -   13. The method of aspect 8, wherein said biological sample         comprises serum, blood, faeces, tissue, a cell, urine and/or         saliva of said human.     -   14. The method of any preceding aspect, wherein said trap,         antibody or fragment is administered by intravenous or         subcutaneous injection and/or is comprised in an injectable         preparation.     -   15. The method of any preceding aspect, wherein the trap,         antibody or fragment is human, eg, a human antibody or antibody         fragment.     -   16. An anti-human TOI trap, antibody or antibody fragment for         use in the method of any preceding aspect, wherein the TOI is         selected from BMP6, ferroportin, haemojuvelin, TMPRSS6 and         hepcidin.

In an embodiment, the trap is a haemojuvelin- or receptor-Fc fusion (ie, a receptor domain fused to an antibody Fc region, eg, as a dimer), wherein the receptor specifically binds to or competes with a TOI is selected from BMP6, ferroportin, haemojuvelin, TMPRSS6 and hepcidin.

-   -   17. The ligand of aspect 17, wherein the trap, antibody or         antibody fragment is fully human.     -   18. The ligand of aspect 16 or 17, wherein the trap, antibody or         antibody fragment is for administration by injection or the         trap, antibody or antibody fragment is provided by an injectable         preparation.

Optionally in any of the configurations, concepts, aspects, embodiments or examples, one or more of the following optional features applies.

Optionally, the ligand (eg, trap, antibody or antibody fragment) is according to one of the following options:—

(i) wherein the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23*04), a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40. (ii) wherein the ligand comprises a VH domain derived from the recombination of human VH segment selected from the group consisting of IGHV3-33*01 and IGHV3-7*01, a human D gene segment and a human JH segment, and wherein said human comprises said selected VH gene segment or the human expresses VH domains derived from the recombination of said selected human VH segment, a human D gene segment and a human JH segment. (iii) wherein the ligand comprises a Vκ domain derived from the recombination of human Vκ segment selected from the group consisting of IGKV3-11*01 and IGKV1-12*01 and a human Jκ segment, and wherein said human comprises said selected Vκ gene segment or the human expresses Vκ domains derived from the recombination of said selected human Vκ segment and a human Jκ segment. (iv) wherein the ligand comprises a Vκ domain derived from the recombination of a human Vκ segment and a human Jκ segment, the human Vκ segment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36 and wherein said human comprises a Vκ gene segment encoding a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the human expresses Vκ domains that comprise a CDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 and wherein said human comprises a Vκ gene segment encoding a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38 or the human expresses Vκ domains that comprise a FW3 comprising a Ser at position 15 shown in SEQ ID NO: 38.

Optionally, instead of a gamma-4 constant region, the ligand (eg, a trap, antibody or fragment) comprises a heavy chain constant region according to any of the following options:—

(v) wherein the ligand comprises a human gamma-1 heavy chain constant region that comprises an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 heavy chain constant regions comprising an Asp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4. (vi) wherein the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6. In an example, the constant region is Fe enhanced, eg, ADCC or CDC enhanced. The skilled person will know techniques (eg, known C region mutations) to enhance ADCC or CDC. (vii) wherein the ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3*01) constant region gene segment and wherein said human comprises (i) said first constant region gene segment (eg, an IGHG3*01), or the human expresses antibodies comprising human gamma-3 heavy chain constant regions encoded by said first human IGHG3 (eg, IGHG3*01) constant region gene segment.

Optionally, the ligand (eg, an antibody or antibody fragment) is according to one of the following options:—

(viii) wherein the ligand comprises a human kappa chain constant region that comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16 and wherein said human comprises (i) an IGKC1*01 human kappa chain constant region gene segment, or the human expresses antibodies comprising human kappa chain constant regions comprising a Val corresponding to position 84 shown in SEQ ID NO: 16 or a Cys at position 87 shown in SEQ ID NO: 16. (ix) wherein the ligand comprises a human IGLC1*01 lambda chain constant region and wherein said human comprises (i) a human IGLC1*01 lambda chain constant region gene segment, or the human expresses antibodies comprising human IGLC1*01 lambda chain constant regions. (x) wherein the ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human epsilon heavy chain constant regions encoded by said first constant region gene segment. (xii) wherein the ligand comprises a human mu heavy chain constant region encoded by a first human mu heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human mu heavy chain constant regions encoded by said first constant region gene segment. (xiii) wherein the ligand comprises a human alpha heavy chain constant region encoded by a first human alpha heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human alpha heavy chain constant regions encoded by said first constant region gene segment. (xiv) wherein the ligand comprises a human delta heavy chain constant region encoded by a first human delta heavy chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human delta heavy chain constant regions encoded by said first constant region gene segment. (xv) wherein the ligand comprises a human kappa light chain constant region encoded by a first human kappa light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human kappa light chain constant regions encoded by said first constant region gene segment. (xvi) wherein the ligand comprises a human lambda light chain constant region encoded by a first human lambda light chain constant region gene segment and wherein said human comprises (i) said first constant region gene segment, or the human expresses antibodies comprising human lambda light chain constant regions encoded by said first constant region gene segment.

Example 9: Nav1.8 (SCN10A; PN3; SNS)

Nav1.8 is a sodium ion channel that in humans is encoded by the SCN10A gene (OMIM: 604427). Nav1.8 is a tetrodotoxin (TTX)-resistant voltage-gated sodium ion channel. It is expressed in the dorsal root ganglion (DRG), in unmyelinated, small-diameter sensory neurons called C-fibres and is involved in the pain pathway (nociception). C-fibres can be activated by noxious thermal or mechanical stimuli and thus can carry pain messages. It is known that Nav1.7, Nav1.8 and Nav1.9 are located in the DRG and play an important role in maintaining chronic inflammatory pain. Nav1.8 a-subunit consists of four homologous domains each with six transmembrane spanning regions of which one is a voltage sensor. Nav1.8 contributes the most to sustaining the depolarising stage of the action potentials in nociceptive sensory neurons by activating quickly and remaining activated after detecting a noxious stimuli. Therefore, Nav1.8 is known to perform a key role in hyperalgesia (increased sensitivity to pain) and allodynia (pain from stimuli that do not usually cause it) which are part of chronic pain. Nav1.8 knockout mice studies have shown that the channel is associated with inflammatory and neuropathic pain. Moreover, Nav1.8 plays a crucial role in cold pain. Mutations in SCN10A are associated to Brugada Syndrome.

Human Nav1.8 polymorphism has been observed. Reference is made to the following, the disclosures of which are incorporated herein by reference in their entirety:—

-   1. Waxman S G et al, Lancet Neurol. 2014 November; 13(11):1152-60.     doi: 10.1016/SI474-4422(14)70150-4; “Sodium channel genes in     pain-related disorders: phenotype-genotype associations and     recommendations for clinical use”. -   2. Hoeijmakers J G et al, “Neurosci Lett. 2014 Dec. 31. pii:     S0304-3940(14)01009-X. doi: 10.1016/j.neulet.2014.12.056; “Painful     peripheral neuropathy and sodium channel mutations”. -   3. Huang J et al, J Neurosci. 2013 Aug. 28; 33(35):14087-97. doi:     10.1523/JNEUROSCI.2710-13.2013; “Small-fiber neuropathy Nav1.8     mutation shifts activation to hyperpolarized potentials and     increases excitability of dorsal root ganglion neurons”. -   4. Han C et al, J Neurol Neurosurg Psychiatry. 2014 May;     85(5):499-505. doi: 10.1136/jnnp-2013-306095. Epub 2013 Sep. 4; “The     G1662S NaV1.8 mutation in small fibre neuropathy: impaired     inactivation underlying DRG neuron hyperexcitability”. -   5. Faber C et al, Proc Natl Acad Sci USA. 2012 Nov. 20;     109(47):19444-9. doi: 10.1073/pnas.1216080109. Epub 2012 Oct. 31;     “Gain-of-function Nav1.8 mutations in painful neuropathy”. -   6. Chambers J C et al, Nat Genet. 2010 February; 42(2):149-52. doi:     10.1038/ng.516. Epub 2010 Jan. 10; “Genetic variation in SCN10A     influences cardiac conduction”. -   7. Arisawa T et al, J Gastroenterol. 2013 January; 48(1):73-80. doi:     10.1007/s00535-012-0602-3. Epub 2012 May 23; “Genetic polymorphisms     of SCN10A are associated with functional dyspepsia in Japanese     subjects”. -   8. van den Boogaard M et al, J Clin Invest. 2014 April;     124(4):1844-52. doi: 10.1172/JC173140. Epub 2014 Mar. 18; “A common     genetic variant within SCN10A modulates cardiac SCN5A expression”. -   9. U.S. Pat. No. 7,582,745: “Compositions and methods for inhibiting     expression of Nav1.8 gene”. -   10. Jabbari J et al, Circ Cardiovasc Genet. 2015 February;     8(1):64-73. doi: 10.1161/HCG.0000000000000022; “Common and Rare     Variants in SCN10A Modulate the Risk of Atrial Fibrillation”.

Mutations in the SCN10A gene account for approximately 5 percent of cases of small fibre neuropathy, a condition characterized by severe pain attacks and a reduced ability to differentiate between hot and cold. The mutations cause amino acid change in the NaV1.8 sodium channel. Many of the mutations result in NaV1.8 sodium channels that open more easily than usual, increasing the flow of sodium ions that produce nerve impulses within nociceptors. This increase in sodium ions enhances transmission of pain signals, causing individuals to be more sensitive to stimulation that might otherwise not cause pain. In this condition, the small fibres that extend from the nociceptors and transmit pain signals (axons) degenerate over time. The cause of this degeneration is unknown, but it likely accounts for signs and symptoms such as the loss of temperature differentiation.

Painful peripheral neuropathies or small-fibre neuropathies are disorders of unmyelinated nociceptive C-fibres causing neuropathic pain and in some cases there is no known cause. A gain-of-function mutation was found in the Nav1.8 encoding SCN10A gene in patients with painful peripheral neuropathy (Faber et al). Faber et al used voltage clamp and current clamp methods along with predictive algorithms on 104 idiopathic patients who did not have the mutation in SCN9A (encoding Nav1.7). They found two gain-of-function mutations in SCN10A in three patients in which both mutations cause increased excitability in DRG sensory neurons and hence they contribute to pain.

Certain common variants (polymorphisms) in the SCN10A gene have been found to increase the risk of developing an irregular heartbeat (arrhythmia). These polymorphisms lead to the production of an altered NaV1.8 sodium channel that can disrupt the electrical signals that control the heartbeat. Specifically, changes in the SCN10A gene are associated with a type of arrhythmia known as heart block. Heart block occurs when the heart's electrical signals are slowed down or interrupted. It is unknown how changes to the NaV1.8 sodium channel lead to heart block.

In an example, the invention provides a method of treating or reducing the risk of a Nav1.8-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.8 protein. The invention also provides a corresponding ligand.

The present invention provides anti-Nav1.8 ligands; and Nav1.8-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of Nav1.8, in particular human Nav1.8 or its ligands and in screening assays to identify other antagonists of Nav1.8 activity. Some of the ligands of the invention are useful for inhibiting Nav1.8-mediated activities.

Anti-Nav1.8 ligands (eg, antibodies and anti-sense RNA) have been developed based on targeting and neutralising so-called “wild-type” human Nav1.8, which is a commonly-occurring form (see, eg, SEQ ID NO: 135). While such ligands may be useful for human patients harbouring this form of human Nav1.8, the inventor considered it useful to investigate the possibility of targeting rarer—but still naturally-occurring—forms of Nav1.8 amongst human populations. In this way, the inventor arrived at insight into the natural occurrences and distributions of rarer human Nav1.8 forms that can serve as useful targets (at the protein or nucleic acid level) for human treatment, prophylaxis and diagnosis pertinent to diseases and conditions mediated or associated with Nav1.8 activity. This particularly provides for tailored therapies, prophylaxis and diagnosis in humans that are devoid of the common Nav1.8 gene or protein.

The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in activity and/or conformation of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to more effectively tailor medicines and diagnosis of patients. The invention, therefore, provides for tailored pharmaceuticals and testing that specifically addresses rarer Nav1.8 polymorphic variant forms. Such forms or “alleles” (at the nucleotide level), comprise one or more changes at the nucleotide and amino acid levels from the corresponding common form nucleotide and amino acids sequences, ie, there are one or more non-synonymous (aka “missense”) changes at the nucleotide level that translate into one or more corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention.

With this realisation, the inventor saw that there is significant industrial and medical application for the invention in terms of guiding the choice of anti-Nav1.8 ligand for administration to human patients for therapy and/or prophylaxis of Nav1.8-mediated or associated diseases or conditions. In this way, the patient receives drugs and ligands that are tailored to their needs—as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example a set of human Nav1.8 variants can be determined on the basis of the following criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population. The inventor selected some variants having at least 3 of the 4 following criteria:—

Naturally-occurring human Nav1.8 variation having a cumulative human allele frequency of 35% or less;

Naturally-occurring human Nav1.8 variation having a total human genotype frequency of about 50% or less;

Naturally-occurring human Nav1.8 variation found in many different human ethnic populations (using the standard categorisation of the 1000 Genomes Project; see Table 2 below); and

Naturally-occurring human Nav1.8 variation found in many individuals distributed across such many different ethnic populations.

The selection can include, as an additional or alternative consideration, selection for nucleotide variation that produced amino acid variation in corresponding Nav1.8 forms (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.

Variants can also be sourced from Ensembl, 1000 Genomes database or the literature.

The inventor identified variants listed in Table 25 below.

TABLE 25 Nav1.8 - Human Polymorphism & SNPs CHROMOSOMAL LOCATION AMINO AMINO (FORWARD NUCLEOTIDE ACID ACID CUMULATIVE EXAMPLE SNP/VARIANT STRAND) CHANGE CHANGE POSITION FREQUENCY¹ CONDITIONS rs138404783 3: 38752313 1661T > C L554P 554 Pain Inflammatory pain Neuropathic pain, eg, SFN 3910G > A A1304T 1304 Pain Inflammatory pain Neuropathic pain, eg, SFN 4984G > A I1706V 1706 Pain Inflammatory pain Neuropathic pain, eg, SFN 2884A > G Dyspepsia EPS DPS Gastric pain 3218C > T Dyspepsia EPS DPS Gastric pain *rs6801957 3: 38725824 C > T 0.305 Brugada Syndrome Cardiac pain Cardiac arrhythmia Heart block Ventricular fibrillation Atrial fibrillation **rs6795970 3: 38725184 G > A A1073V 1073 0.264 Cardiac pain Cardiac arrhythmia Heart block Ventricular fibrillation Atrial fibrillation G1662S 1662 Pain Inflammatory pain Neuropathic pain, eg, SFN ***rs10428132 3: 38736063 G > T 0.296 Brugada Syndrome Cardiac pain Cardiac arrhythmia Heart block Ventricular fibrillation Atrial fibrillation **** rs12632942 3: 38723507 A > G L1092P 1092 0.237 Pain Inflammatory pain Neuropathic pain, eg, SFN Brugada Syndrome Cardiac pain Cardiac arrhythmia Heart block Ventricular fibrillation Atrial fibrillation ***** rs57326399 3: 38726809 T > C I962V 962 0.231 Pain Inflammatory pain Neuropathic pain, eg, SFN Brugada Syndrome Cardiac pain Cardiac arrhythmia Heart block Ventricular fibrillation Atrial fibrillation + rs7630989 3: 38752449 A > G S509P 509 0.137 Pain Inflammatory pain Neuropathic pain, eg, SFN Brugada Syndrome Cardiac pain Cardiac arrhythmia Heart block Ventricular fibrillation Atrial fibrillation ++ rs74717885 3: 38763578 T > C I206M 206 0.048 Pain Inflammatory pain Neuropathic pain, eg, SFN Brugada Syndrome Cardiac pain Cardiac arrhythmia Heart block Ventricular fibrillation Atrial fibrillation +++ rs144270136 3: 38793743 G > A R90W 90 0.009 Neuropathic pain, eg, SFN Brugada Syndrome Cardiac pain Cardiac arrhythmia Heart block Ventricular fibrillation Atrial fibrillation SFN = small fibre neuropathy or pain associated with small fibre neuropathy EPS = Epigastric pain syndrome PDS = Postprandial distress syndrome ¹According to Ensembl, dbSNP *rs6801957 is present in humans at an average cumulative frequency of 31% according to the 1000 Genomes Phase I database, but is higher in AMR, EUR, CLM, MXL, PUR, CEU, FIN, GBR, IBS and TSI populations; thus in an embodiment, in the present invention involving Nav1.8 the human is of AMR ancestry, eg, of CLM, MXL or PUR ancestry; or of EUR ancestry, eg, of CEU, FIN, GBR, IBS or TSI ancestry. **rs6795970 is present in humans at an average cumulative frequency of 26% according to the 1000 Genomes Phase I database, but is higher in AMR, EUR, CLM, MXL, PUR, CEU, FIN, GBR, IBS and TSI populations; thus in an embodiment, in the present invention involving Nav1.8 the human is of AMR ancestry, eg, of CLM, MXL or PUR ancestry; or of EUR ancestry, eg, of CEU, FIN, GBR, IBS or TSI ancestry. ***rs10428132 is present in humans at an average cumulative frequency of 30% according to the 1000 Genomes Phase I database, but is higher in AMR, EUR, CLM, MXL, PUR, CEU, FIN, GBR, IBS and TSI populations; thus in an embodiment, in the present invention involving Nav1.8 the human is of AMR ancestry, eg, of CLM, MXL or PUR ancestry; or of EUR ancestry, eg, of CEU, FIN, GBR, IBS or TSI ancestry. **** rs12632942 is present in humans at an average cumulative frequency of 24% according to the 1000 Genomes Phase I database, but is higher in ASN, EUR, CHB, CHS, JPT, IBS and TSI populations; thus in an embodiment, in the present invention involving Nav1.8 the human is of ASN ancestry, eg, of CHB, CHS or JPT ancestry; or of EUR ancestry, eg, of IBS or TSI ancestry. ***** rs57326399 is present in humans at an average cumulative frequency of 23% according to the 1000 Genomes Phase I database, but is higher in ASN, EUR, CHB, CHS, JPT, CEU, GBR, IBS and TSI populations; thus in an embodiment, in the present invention involving Nav1.8 the human is of ASN ancestry, eg, of CHB, CHS or JPT ancestry; or of EUR ancestry, eg, of CEU, GBR, IBS or TSI ancestry. + - rs7630989 is present in humans at an average cumulative frequency of 14% according to the 1000 Genomes Phase I database, but is higher in AFR, ASN, ASW, LWK, YRI, CHB, CHS and JPT populations; thus in an embodiment, in the present invention involving Nav1.8 the human is of ASN ancestry, eg, of CHB, CHS or JPT ancestry; or of EUR ancestry, eg, of CEU, GBR, IBS or TSI ancestry. ++ - rs74717885 is present in humans at an average cumulative frequency of 5% according to the 1000 Genomes Phase I database, but is higher in ASN, CHB, CHS and JPT populations; thus in an embodiment, in the present invention involving Nav1.8 the human is of ASN ancestry, eg, of CHB, CHS or JPT ancestry. +++ - rs144270136 is present in humans at an average cumulative frequency of 5% according to the 1000 Genomes Phase I database, but is higher in ASN, CHB, CHS and JPT populations; thus in an embodiment, in the present invention involving Nav1.8 the human is of ASN ancestry, eg, of CHB, CHS or JPT ancestry. In an embodiment, variations are with reference to transcript ENST00000449082 (the sequence of this transcript being incorporated herein in its entirety).

Optionally for any mutation, the condition is a condition listed in Table 25.

In an embodiment, the human comprises a SNP selected from the SNPs in column 1 of Table 25; optionally also the ligand specifically binds a human Nav1.8 encoded by a nucleotide sequence comprising said SNP.

In an embodiment, the human comprises a Nav1.8 amino acid variation selected from the variations in column 4 of Table 25; optionally also the ligand specifically binds a human Nav1.8 comprising said variation.

In an example, the invention provides a method of treating or reducing the risk of a Nav1.8-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain). These amino acid variations are found in naturally-occurring Nav1.8 variants in humans. Said human comprises a nucleotide sequence encoding a Nav1.8 protein comprising said selected amino acid.

In an example, the selected amino acid is L554P. In an example, the selected amino acid is A1304T. In an example, the selected amino acid is I1706V. In an example, the selected amino acid is G1662S.

The disease or condition is, for example, any disease or condition described above in the Example relating to Nav1.7 and the disclosure of such diseases and conditions are hereby also imported into this example to provide for possible combination of the present invention relating to Nav1.8 (eg, for use in one or more claims herein). In one embodiment, the condition is a condition listed in Table 25 or a symptom of such a condition. In an example, the condition is pain or a symptom of pain, eg, a symptom of neuropathic pain. In an example, the condition is selected from the group consisting of paroxysmal pain (eg, of hands and/or feet), hand pain, foot pain, limb pain (eg, leg pain), cramp (eg, foot cramp or leg cramp), tingling (e, foot, hand or leg tingling) and pain associated with clothing contact with the human. In an example, the condition is an abnormal threshold for warmth or cold sensation (eg, in a foot or feet). In an example, the condition is joint pain. In an example, the condition is dry eye sensation, diahrrhoea, urinary condition, heart palpitations or dizziness. In an example, the condition is insensitivity to pain. In an example, the condition is episodic pain (eg, familial episodic pain).

In one embodiment, the pain is chronic pain.

In one embodiment, the pain is neuropathic pain, eg, chronic neuropathic pain, eg, peripheral neuropathic pain. For example, the pain is painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN) or trigeminal neuralgia (TN).

In an example, the pain is spinal cord injury pain, multiple sclerosis pain, phantom limb pain, post-stroke pain, chronic back pain, osteoarthritis pain, cancer-associated pain or HIV-associated pain.

In one embodiment, the pain is inflammatory pain, eg, chronic inflammatory pain.

In an example, the ligand of the invention comprises an anti-human Nav1.8 binding site, wherein the binding site is a human or humanized binding site, eg, the binding site comprises or consists of a human or humanized antibody variable domain or plurality of variable domains (eg, human VH/VL binding site(s)). Additionally or alternatively, the ligand comprises one or more human antibody constant regions (eg, a human antibody CH1, CH2, CH3 (or all of these) or Fc). In an example, the ligand is an antibody that comprises human or humanized variable regions and human constant regions (eg, bearing one or more mutations to enhance or dampen Fc function in a human patient).

An example provides a ligand (eg, an antibody or antibody fragment) for treating or reducing the risk of a Nav1.8-mediated disease or condition in a human in need thereof, the method comprising administering to said human said ligand, wherein the ligand specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain). Said human comprises a nucleotide sequence encoding a Nav1.8 protein comprising said selected amino acid.

In an example, the invention provides a method of targeting Nav1.8 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain). Said human comprises a nucleotide sequence encoding a Nav1.8 protein comprising said selected amino acid. In an example, the human is suffering from or at risk of a Nav1.8-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.8-mediated disease or condition in the human.

An example also provides a ligand (eg, an antibody or antibody fragment) for targeting Nav1.8 in a human, the method comprising administering to said human said ligand, wherein the ligand specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain). Said human comprises a nucleotide sequence encoding a (eg, said) Nav1.8 protein comprising said selected amino acid. In an example, the human is suffering from or at risk of a Nav1.8-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.8-mediated disease or condition in the human.

In an embodiment, (i) the antibody or fragment comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

Additionally or alternatively, in an embodiment, (i) the antibody or fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an example, the ligand, antibody or fragment is for treating or reducing the risk of (or treats or reduces the risk of) pain or itching, optionally wherein the antibody is a humanized mouse or Camelid (eg, llama or camel) antibody.

In a specific embodiment, the anti-Nav1.8 ligand, antibody or fragment also specifically binds to another Nav selected from Nav1.1-1.9, eg, it specifically binds to Nav1.7 or 1.9.

In a specific embodiment, the anti-Nav1.8 ligand, antibody or fragment of present invention comprises an Fc region as per the disclosure in the Nav1.7 example above.

The ligand, antibody or fragment according to the invention is for treating or preventing or reducing the risk of (or treats or prevents or reduces the risk of), for example, any disease or condition disclosed in WO2011/051351 or U.S. Pat. No. 7,582,745, the disclosures of which diseases and conditions are incorporated herein by reference for potential inclusion in one or more claims herein. Guidance on obtaining and testing antibodies can also be found in that PCT application.

Further encompassed by the invention is the use of the ligand, antibody or fragment in the manufacture of a medicament for use to attenuate or inhibit a Nav1.8-mediated disease or disorder in a human. Nav1.8-mediated or related disorders which are treated by the ligand, antibody or fragment of the invention include, for example, a pain or itching disease or condition.

Thus, a ligand, antibody or fragment of the invention is useful as a therapeutic agent in the treatment of a condition involving Nav1.8 expression and/or activity. One embodiment, among others, is a method of treatment comprising administering an effective amount of a ligand, antibody or fragment of the invention to a patient in need thereof, wherein functional consequences of Nav1.8 activation are decreased. Another embodiment, among others, is a method of treatment comprising (i) identifying a patient demonstrating Nav1.8 expression or activity, and (ii) administering an effective amount of a ligand, antibody or fragment of the invention to the patient, wherein a functional consequence of Nav1.8 activation are attenuated. An effective amount according to the invention is an amount that modulates (e.g. decreases) the functional consequences of Nav1.8 activation so as to modulate (e.g. decrease or lessen) the severity of at least one symptom of the particular disease or disorder being treated, but not necessarily cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, comprising administering to a patient in need thereof an effective amount of one or more ligands, antibodies or fragments of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein such that the severity of at least one symptom of any of the disorders is reduced. Another embodiment of the invention, among others, is a method of antagonizing at least one effect of Nav1.8 comprising contacting with or administering an effective amount of one or more ligands, antibodies or fragments of the present invention such that said at least one effect of Nav1.8 is antagonized, e.g. the ability of Nav1.8 to form an ion channel, such as a sodium channel.

Tailoring Antibodies to Rarer Nav1.8 Variant Profile

As outlined herein (for example, in the context of PCSK9 in Example 1 or in the context of the Nav1.7 Example herein), the invention includes the possibility to tailor treatment of humans further by selecting antibody-based ligands with variable domains and/or constant domains based on gene segments found in many humans of the ethnic populations where the variant TOI forms are found to meet the selection criteria of the invention. This also applies mutatis mutandis where the TOI is human Nav1.8, as in the present example. Thus, all disclosure herein relating to tailoring variable and/or constant domains apply to the present example, relating to Nav1.8 and is combinable for use in one or more claims herein. All disclosure relating to variable and constant regions and polymorphism under the subtitle “Tailoring Antibodies to Rarer Nav1.7 Variant Profile” in the Nav1.7 Example is also incorporated into the present Nav1.8 Example for use mutatis mutandis with the present Nav1.8 invention and for possible inclusion in Nav1.8-limited claims herein.

Determination of Specific Binding of Ligands of the Invention to NAV1.8 Variants

The specific binding of ligands of the invention to Nav1.8 variants can be performed using the SPR method described in Example 1.

Embodiments are provided as follows:—

Methods with VH Tailoring

-   1. A method of treating or reducing the risk of a Nav1.8-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a human Nav1.8 protein     that comprises an amino acid selected from the group consisting of     L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M     and R90W or an amino acid encoded by a SNP selected from the group     consisting of rs6801957, rs6795970, rs10428132, rs12632942,     rs57326399, rs7630989, rs74717885 and rs144270136; optionally     selected from the group consisting of L554P, A1304T, I1706V and     G1662S (eg, when the condition is neuropathic pain);

wherein (i) the ligand comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an alternative, clause 1 provides:—

A method of targeting Nav1.8 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain); wherein (i) the ligand comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an example, the human is suffering from or at risk of a Nav1.8-mediated disease or condition.

In an example, the method treats or reduces the risk of a Nav1.8-mediated disease or condition in the human.

-   2. The method of clause 1, wherein said amino acid is selected from     the group consisting of L554P, A1304T, I1706V and G1662S; optionally     wherein said disease or condition is a pain disease or condition,     eg, inflammatory pain or neuropathic pain, (eg, SFN). -   3. The method of clause 1, wherein said amino acid is selected from     the group consisting of 2884A>G and 3218C>T; optionally wherein said     disease or condition is pain or is selected from dyspepsia, EPS, DPS     and gastric pain. -   4. The method of clause 1, wherein said amino acid is encoded by a     SNP selected from the group consisting of rs6801957, rs6795970 and     rs10428132; optionally wherein said disease or condition is selected     from the group consisting of Brugada syndrome, cardiac pain, cardiac     arrhythmia, heart block, ventricular fibrillation and atrial     fibrillation. -   5. The method of any preceding clause, wherein the VH gene segment     comprised by said human is a germline VH gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.8     protein comprising said selected amino acid and/or a Nav1.8 protein     comprising said selected amino acid. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes a Nav1.8 protein comprising said selected amino acid     and/or (b) a Nav1.8 protein comprising said selected amino acid,     optionally, wherein the determining step is performed before     administration of the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.8 protein comprising said selected amino     acid. -   10. The method of clause 9, wherein the assaying comprises

contacting the biological sample with at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides of a nucleotide sequence encoding a Nav1.8 protein comprising said selected amino acid or comprising an antisense sequence of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises a nucleotide sequence encoding said selected amino acid, thereby forming a complex when at least one nucleotide sequence encoding the Nav1.8 protein comprising said selected amino acid is present; and detecting the presence or absence of the complex, wherein detecting the presence of the complex determines that the human comprises the Nav1.8 protein comprising said selected amino acid.

-   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.8 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 136 or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.8 protein comprising     said selected amino acid. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The method of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The method of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs138404783, rs6801957, rs6795970, rs10428132,     rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136. -   21. The method of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is administered by inhaled,     intravenous or subcutaneous administration and/or is comprised in an     inhalable or injectable preparation.

Ligands with VH Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of a Nav1.8-mediated disease     or condition (eg, asthma) in a human in need thereof, wherein the     ligand specifically binds a human Nav1.8 protein that comprises an     amino acid selected from the group consisting of L554P, A1304T,     I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an     amino acid encoded by a SNP selected from the group consisting of     rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,     rs74717885 and rs144270136; optionally selected from the group     consisting of L554P, A1304T, I1706V and G1662S (eg, when the     condition is neuropathic pain);

wherein (i) the ligand comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting Nav1.8 in a human, wherein the ligand specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain);

wherein (i) the ligand comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain). In an example, the human is suffering from or at risk of a Nav1.8-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.8-mediated disease or condition in the human.

-   2. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of L554P, A1304T, I1706V and     G1662S; optionally wherein said disease or condition is a pain     disease or condition, eg, inflammatory pain or neuropathic pain,     (eg, SFN). -   3. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of 2884A>G and 3218C>T;     optionally wherein said disease or condition is pain or is selected     from dyspepsia, EPS, DPS and gastric pain. -   4. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of rs6801957, rs6795970 and     rs10428132; optionally wherein said disease or condition is selected     from the group consisting of Brugada syndrome, cardiac pain, cardiac     arrhythmia, heart block, ventricular fibrillation and atrial     fibrillation. -   5. The ligand of any preceding clause, wherein the VH gene segment     comprised by said human is a germline VH gene segment. -   6. The ligand of any preceding clause said method comprising, before     said administering, selecting a human comprising said nucleotide     sequence of (ii), wherein the human is the human of clause 1. -   7. The ligand of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.8     protein comprising said selected amino acid selected and/or a Nav1.8     protein comprising said selected amino acid. -   8. The ligand of any preceding clause, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes a Nav1.8 protein comprising said selected     amino acid and/or (b) a Nav1.8 protein comprising said selected     amino acid, optionally, wherein the determining step is performed     before administration of the antibody to the human. -   9. The ligand of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.8 protein comprising said selected amino     acid. -   10. The ligand of clause 9, wherein the assaying comprises     contacting the biological sample with at least one oligonucleotide     probe comprising a sequence of at least 10 contiguous nucleotides of     a nucleotide sequence encoding a Nav1.8 protein comprising said     selected amino acid or comprising an antisense sequence of said     contiguous nucleotides, wherein said sequence of contiguous     nucleotides comprises a nucleotide sequence encoding said selected     amino acid, thereby forming a complex when at least one nucleotide     sequence encoding the Nav1.8 protein comprising said selected amino     acid is present; and detecting the presence or absence of the     complex, wherein detecting the presence of the complex determines     that the human comprises the Nav1.8 protein comprising said selected     amino acid. -   11. The ligand of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The ligand of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.8 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 136, or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.8 protein comprising     said selected amino acid. -   14. The ligand of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The ligand of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The ligand of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The ligand of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The ligand of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The ligand of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The ligand of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs138404783, rs6801957, rs6795970, rs10428132,     rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136. -   21. The ligand of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is for inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation.

Methods with Gamma-4 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.8-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a human Nav1.8 protein     that comprises an amino acid selected from the group consisting of     L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M     and R90W or an amino acid encoded by a SNP selected from the group     consisting of rs6801957, rs6795970, rs10428132, rs12632942,     rs57326399, rs7630989, rs74717885 and rs144270136; optionally     selected from the group consisting of L554P, A1304T, I1706V and     G1662S (eg, when the condition is neuropathic pain);

wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an alternative, clause 1 provides:—

A method of targeting Nav1.8 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain); wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an example, the human is suffering from or at risk of a Nav1.8-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.8-mediated disease or condition in the human.

-   2. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of L554P, A1304T, I1706V and     G1662S; optionally wherein said disease or condition is a pain     disease or condition, eg, inflammatory pain or neuropathic pain,     (eg, SFN). -   3. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of 2884A>G and 3218C>T;     optionally wherein said disease or condition is pain or is selected     from dyspepsia, EPS, DPS and gastric pain. -   4. The method of any preceding clause, wherein said amino acid is     encoded by a SNP selected from the group consisting of rs6801957,     rs6795970 and rs10428132; optionally wherein said disease or     condition is selected from the group consisting of Brugada syndrome,     cardiac pain, cardiac arrhythmia, heart block, ventricular     fibrillation and atrial fibrillation. -   5. The method of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.8     protein comprising said selected amino acid and/or a Nav1.8 protein     comprising said selected amino acid. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes a Nav1.8 protein comprising said selected amino acid     and/or (b) a Nav1.8 protein comprising said selected amino acid,     optionally, wherein the determining step is performed before     administration of the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.8 protein comprising said selected amino     acid. -   10. The method of clause 9, wherein the assaying comprises

contacting the biological sample with at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides of a nucleotide sequence encoding a Nav1.8 protein comprising said selected amino acid or comprising an antisense sequence of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises a nucleotide sequence encoding said selected amino acid, thereby forming a complex when at least one nucleotide sequence encoding the Nav1.8 protein comprising said selected amino acid is present; and

detecting the presence or absence of the complex, wherein detecting the presence of the complex determines that the human comprises the Nav1.8 protein comprising said selected amino acid.

-   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.8 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 136 or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.8 protein comprising     said selected amino acid. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The method of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The method of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting rs138404783, rs6801957, rs6795970, rs10428132,     rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136. -   21. The method of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is administered by inhaled,     intravenous or subcutaneous administration and/or is comprised in an     inhalable or injectable preparation. -   22. The method of any preceding clause, wherein the human gamma-4     heavy chain constant region of the ligand comprises the amino acid     sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof. -   23. The method of any preceding clause, wherein the human gamma-4     heavy chain constant region comprises 228P and 235E.

Ligands with Gamma-4 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of a Nav1.8-mediated disease     or condition (eg, pain) in a human in need thereof, wherein the     ligand specifically binds a human Nav1.8 protein that comprises an     amino acid selected from the group consisting of L554P, A1304T,     I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an     amino acid encoded by a SNP selected from the group consisting of     rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,     rs74717885 and rs144270136; optionally selected from the group     consisting of L554P, A1304T, I1706V and G1662S (eg, when the     condition is neuropathic pain);

wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting Nav1.8 in a human, wherein the ligand specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain);

wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an example, the human is suffering from or at risk of a Nav1.8-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.8-mediated disease or condition in the human.

-   2. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of L554P, A1304T, I1706V and     G1662S; optionally wherein said disease or condition is a pain     disease or condition, eg, inflammatory pain or neuropathic pain,     (eg, SFN). -   3. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of 2884A>G and 3218C>T;     optionally wherein said disease or condition is pain or is selected     from dyspepsia, EPS, DPS and gastric pain. -   4. The ligand of any preceding clause, wherein said amino acid is     encoded by a SNP selected from the group consisting of rs6801957,     rs6795970 and rs10428132; optionally wherein said disease or     condition is selected from the group consisting of Brugada syndrome,     cardiac pain, cardiac arrhythmia, heart block, ventricular     fibrillation and atrial fibrillation. -   5. The ligand of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The ligand of any preceding clause said method comprising, before     said administering, selecting a human comprising said nucleotide     sequence of (ii), wherein the human is the human of clause 1. -   7. The ligand of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.8     protein comprising said selected amino acid selected and/or a Nav1.8     protein comprising said selected amino acid. -   8. The ligand of any preceding clause, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes a Nav1.8 protein comprising said selected     amino acid and/or (b) a Nav1.8 protein comprising said selected     amino acid, optionally, wherein the determining step is performed     before administration of the antibody to the human. -   9. The ligand of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.8 protein comprising said selected amino     acid. -   10. The ligand of clause 9, wherein the assaying comprises

contacting the biological sample with at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides of a nucleotide sequence encoding a Nav1.8 protein comprising said selected amino acid or comprising an antisense sequence of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises a nucleotide sequence encoding said selected amino acid, thereby forming a complex when at least one nucleotide sequence encoding the Nav1.8 protein comprising said selected amino acid is present; and

detecting the presence or absence of the complex, wherein detecting the presence of the complex determines that the human comprises the Nav1.8 protein comprising said selected amino acid.

-   11. The ligand of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The ligand of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.8 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 136, or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.8 protein comprising     said selected amino acid. -   14. The ligand of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The ligand of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The ligand of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The ligand of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The ligand of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The ligand of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The ligand of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs138404783, rs6801957, rs6795970, rs10428132,     rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136. -   21. The ligand of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is for inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation. -   22. The ligand of any preceding clause, wherein the human gamma-4     heavy chain constant region of the ligand comprises the amino acid     sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof. -   23. The ligand of any preceding clause, wherein the human gamma-4     heavy chain constant region comprises 228P and 235E.

Methods with Gamma-2 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.8-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a human Nav1.8 protein     that comprises an amino acid selected from the group consisting of     L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M     and R90W or an amino acid encoded by a SNP selected from the group     consisting of rs6801957, rs6795970, rs10428132, rs12632942,     rs57326399, rs7630989, rs74717885 and rs144270136; optionally     selected from the group consisting of L554P, A1304T, I1706V and     G1662S (eg, when the condition is neuropathic pain);

wherein (i) the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an alternative, clause 1 provides:—

A method of targeting Nav1.8 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain); wherein (i) the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and

wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an example, the human is suffering from or at risk of a Nav1.8-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.8-mediated disease or condition in the human.

-   2. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of L554P, A1304T, I1706V and     G1662S; optionally wherein said disease or condition is a pain     disease or condition, eg, inflammatory pain or neuropathic pain,     (eg, SFN). -   3. The method of any preceding clause, wherein said amino acid is     selected from the group consisting of 2884A>G and 3218C>T;     optionally wherein said disease or condition is pain or is selected     from dyspepsia, EPS, DPS and gastric pain. -   4. The method of any preceding clause, wherein said amino acid is     encoded by a SNP selected from the group consisting of rs6801957,     rs6795970 and rs10428132; optionally wherein said disease or     condition is selected from the group consisting of Brugada syndrome,     cardiac pain, cardiac arrhythmia, heart block, ventricular     fibrillation and atrial fibrillation. -   5. The method of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.8     protein comprising said selected amino acid and/or a Nav1.8 protein     comprising said selected amino acid. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes a Nav1.8 protein comprising said selected amino acid     and/or (b) a Nav1.8 protein comprising said selected amino acid,     optionally, wherein the determining step is performed before     administration of the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.8 protein comprising said selected amino     acid. -   10. The method of clause 9, wherein the assaying comprises

contacting the biological sample with at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides of a nucleotide sequence encoding a Nav1.8 protein comprising said selected amino acid or comprising an antisense sequence of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises a nucleotide sequence encoding said selected amino acid, thereby forming a complex when at least one nucleotide sequence encoding the Nav1.8 protein comprising said selected amino acid is present; and detecting the presence or absence of the complex, wherein detecting the presence of the complex determines that the human comprises the Nav1.8 protein comprising said selected amino acid.

-   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.8 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 136 or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.8 protein comprising     said selected amino acid. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The method of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The method of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting rs138404783, rs6801957, rs6795970, rs10428132,     rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136. -   21. The method of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is administered by inhaled,     intravenous or subcutaneous administration and/or is comprised in an     inhalable or injectable preparation. -   22. The method of any preceding clause, wherein the human gamma-2     heavy chain constant region of the ligand comprises IGHG2*01 amino     acid sequence or an ADCC inactivated version thereof.

Ligands with Gamma-2 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of a Nav1.8-mediated disease     or condition (eg, pain) in a human in need thereof, wherein the     ligand specifically binds a human Nav1.8 protein that comprises an     amino acid selected from the group consisting of L554P, A1304T,     I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an     amino acid encoded by a SNP selected from the group consisting of     rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,     rs74717885 and rs144270136; optionally selected from the group     consisting of L554P, A1304T, I1706V and G1662S (eg, when the     condition is neuropathic pain);

wherein (i) the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and

wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting Nav1.8 in a human, wherein the ligand specifically binds a human Nav1.8 protein that comprises an amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain);

wherein (i) the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.8 protein comprising said amino acid selected from the group consisting of L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W or an amino acid encoded by a SNP selected from the group consisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136; optionally selected from the group consisting of L554P, A1304T, I1706V and G1662S (eg, when the condition is neuropathic pain).

In an example, the human is suffering from or at risk of a Nav1.8-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.8-mediated disease or condition in the human.

-   2. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of L554P, A1304T, I1706V and     G1662S; optionally wherein said disease or condition is a pain     disease or condition, eg, inflammatory pain or neuropathic pain,     (eg, SFN). -   3. The ligand of any preceding clause, wherein said amino acid is     selected from the group consisting of 2884A>G and 3218C>T;     optionally wherein said disease or condition is pain or is selected     from dyspepsia, EPS, DPS and gastric pain. -   4. The ligand of any preceding clause, wherein said amino acid is     encoded by a SNP selected from the group consisting of rs6801957,     rs6795970 and rs10428132; optionally wherein said disease or     condition is selected from the group consisting of Brugada syndrome,     cardiac pain, cardiac arrhythmia, heart block, ventricular     fibrillation and atrial fibrillation. -   5. The ligand of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The ligand of any preceding clause said method comprising, before     said administering, selecting a human comprising said nucleotide     sequence of (ii), wherein the human is the human of clause 1. -   7. The ligand of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.8     protein comprising said selected amino acid selected and/or a Nav1.8     protein comprising said selected amino acid. -   8. The ligand of any preceding clause, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes a Nav1.8 protein comprising said selected     amino acid and/or (b) a Nav1.8 protein comprising said selected     amino acid, optionally, wherein the determining step is performed     before administration of the antibody to the human. -   9. The ligand of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.8 protein comprising said selected amino     acid. -   10. The ligand of clause 9, wherein the assaying comprises

contacting the biological sample with at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides of a nucleotide sequence encoding a Nav1.8 protein comprising said selected amino acid or comprising an antisense sequence of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises a nucleotide sequence encoding said selected amino acid, thereby forming a complex when at least one nucleotide sequence encoding the Nav1.8 protein comprising said selected amino acid is present; and detecting the presence or absence of the complex, wherein detecting the presence of the complex determines that the human comprises the Nav1.8 protein comprising said selected amino acid.

-   11. The ligand of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The ligand of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.8 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 136, or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.8 protein comprising     said selected amino acid. -   14. The ligand of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The ligand of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The ligand of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The ligand of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The ligand of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The ligand of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The ligand of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs138404783, rs6801957, rs6795970, rs10428132,     rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136. -   21. The ligand of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is for inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation. -   22. The ligand of any preceding clause, wherein the human gamma-2     heavy chain constant region of the ligand comprises IGHG2*01 amino     acid sequence or an ADCC inactivated version thereof.

In any embodiment or example of the Nav1.8-focused invention, the method optionally reduces a symptom of neuropathic pain or inflammatory pain; or optionally the ligand is for reducing a symptom of neuropathic pain or inflammatory pain.

In any embodiment or example of the Nav1.8-focused invention said selected amino acid may be regarded as a mutation in SEQ ID NO: 135.

In an example, the human is suffering from or at risk of a Nav1.8-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.8-mediated disease or condition in the human.

Example 10: Nav1.9 (SCN11A; NaN; SCN12A; SNS-2)

Nav1.9 (Sodium channel, voltage-gated, type XI, alpha subunit) is a voltage-gated sodium ion channel protein which in humans is encoded by the SCN11 A gene (OMIM: 604385). Nav1.9 has high expression levels in peripheral sensory neurons and is thought to carry the NaN current, a persistent and tetrodrotoxin (TTX)-resistant voltage-gated Na Current, involved in pain-related signalling. Nav1.9 is found within small sensory neurons (<30 m diameter) of dorsal root ganglia (DRG) and trigeminal ganglia. Nav1.9 is relatively resistant to TTX, but has kinetic properties distinct from the similarly TTX-resistant Nav1.8, producing a persistent current with an activation potential of −70 mV, close to the resting membrane potential, where it may set the threshold for activation. A gain-of-function mutation of SCN11A was reported to be linked to congenital insensitivity to pain (Leipold et al 2013), familial episodic pain (Zhang et al 2013) and painful peripheral neuropathy (Huang et al 2014).

Human Nav1.9 polymorphism has been observed. Reference is made to the following, the disclosures of which are incorporated herein by reference in their entirety:—

1. Waxman S G et al, Lancet Neurol. 2014 November; 13(11):1152-60. doi: 10.1016/S1474-4422(14)70150-4; “Sodium channel genes in pain-related disorders: phenotype-genotype associations and recommendations for clinical use”.

-   2. Hoeijmakers J G et al, “Neurosci Lett. 2014 Dec. 31. pii:     S0304-3940(14)01009-X. doi: 10.1016/j.neulet.2014.12.056; “Painful     peripheral neuropathy and sodium channel mutations”. -   3. Huang J et al, Brain. 2014 June; 137(Pt 6):1627-42. doi:     10.1093/brain/awu079. Epub 2014 Apr. 27; “Gain-of-function mutations     in sodium channel Na(v)1.9 in painful neuropathy”. -   4. Leipold E et al, Nat Genet. 2013 November; 45(11):1399-404. doi:     10.1038/ng.2767. Epub 2013 Sep. 15; “A de novo gain-of-function     mutation in SCN11A causes loss of pain perception”. -   5. Zhang X Y et al, Am J Hum Genet. 2013 Nov. 7; 93(5):957-66. doi:     10.1016/j.ajhg.2013.09.016. Epub 2013 Oct. 24; “Gain-of-function     mutations in SCN11A cause familial episodic pain”. -   6. Bennett D L, Brain. 2014 June; 137(Pt 6):1574-6. doi:     10.1093/brain/awu105. Epub 2014 Apr. 27; “Voltage-gated sodium     channel mutations and painful neuropathy: Na(v)1.9 joins the     family”.

In an example, the invention provides a method of treating or reducing the risk of a Nav1.9-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.9 protein. The invention also provides a corresponding ligand.

The present invention provides anti-Nav1.9 ligands; and Nav1.9-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of Nav1.9, in particular human Nav1.9 or its ligands and in screening assays to identify other antagonists of Nav1.9 activity. Some of the ligands of the invention are useful for inhibiting Nav1.9-mediated activities.

Anti-Nav1.9 ligands (eg, antibodies and anti-sense RNA) have been developed based on targeting and neutralising so-called “wild-type” human Nav1.9, which is a commonly-occurring form (see, eg, SEQ ID NO: 137). While such ligand may be useful for human patients harbouring this form of human Nav1.9, the inventor considered it useful to investigate the possibility of targeting rarer—but still naturally-occurring—forms of Nav1.9 amongst human populations. In this way, the inventor arrived at insight into the natural occurrences and distributions of rarer human Nav1.9 forms that can serve as useful targets (at the protein or nucleic acid level) for human treatment, prophylaxis and diagnosis pertinent to diseases and conditions mediated or associated with Nav1.9 activity. This particularly provides for tailored therapies, prophylaxis and diagnosis in humans that are devoid of the common Nav1.9 gene or protein.

The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in activity and/or conformation of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to more effectively tailor medicines and diagnosis of patients. The invention, therefore, provides for tailored pharmaceuticals and testing that specifically addresses rarer Nav1.9 polymorphic variant forms. Such forms or “alleles” (at the nucleotide level), comprise one or more changes at the nucleotide and amino acid levels from the corresponding common form nucleotide and amino acids sequences, ie, there are one or more non-synonymous (aka “missense”) changes at the nucleotide level that translate into one or more corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention.

With this realisation, the inventor saw that there is significant industrial and medical application for the invention in terms of guiding the choice of anti-Nav1.9 ligand for administration to human patients for therapy and/or prophylaxis of Nav1.9-mediated or associated diseases or conditions. In this way, the patient receives drugs and ligands that are tailored to their needs—as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example a set of human Nav1.9 variants can be determined on the basis of the following criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population. The inventor selected some variants having at least 3 of the 4 following criteria:—

Naturally-occurring human Nav1.9 variation having a cumulative human allele frequency of 35% or less;

Naturally-occurring human Nav1.9 variation having a total human genotype frequency of about 50% or less;

Naturally-occurring human Nav1.9 variation found in many different human ethnic populations (using the standard categorisation of the 1000 Genomes Project; see Table 2 below); and

Naturally-occurring human Nav1.9 variation found in many individuals distributed across such many different ethnic populations.

The selection can include, as an additional or alternative consideration, selection for nucleotide variation that produced amino acid variation in corresponding Nav1.9 forms (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.

Variants can also be sourced from Ensembl, 1000 Genomes database or the literature.

The inventor identified variants listed in Table 26 below.

TABLE 26 Nav1.9 - Human Polymorphism & SNPs CHROMOSOMAL LOCATION AMINO (FORWARD NUCLEOTIDE ACID AMINO ACID CUMULATIVE EXAMPLE SNP/VARIANT STRAND) CHANGE CHANGE POSITION FREQUENCY¹ CONDITIONS 1142T > C I381T 381 SFN Numbness (eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feet or legs) Joint pain Decreased vibration sensitivity Abnormal warmth or cold sensation K419N 419 SFN Numbness (eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feet or legs) Joint pain Decreased vibration sensitivity Abnormal warmth or cold sensation A582T 582 SFN Numbness (eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feet or legs) Joint pain Decreased vibration sensitivity Abnormal warmth or cold sensation A681D 681 SFN Numbness (eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feet or legs) Joint pain Decreased vibration sensitivity Abnormal warmth or cold sensation A842P 842 SFN Numbness (eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feet or legs) Joint pain Decreased vibration sensitivity Abnormal warmth or cold sensation 3473T > C L1158P 1158 SFN Numbness (eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feet or legs) Joint pain Decreased vibration sensitivity Abnormal warmth or cold sensation F1689L 1689 SFN Numbness (eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feet or legs) Joint pain Decreased vibration sensitivity Abnormal warmth or cold sensation 2432T>C L811P 811 Pain insensitivity Abnormal warmth or cold sensation 673C > T R225C 225 Pain Episodic pain (eg, familial episodic pain) 2423C > G A808G 808 Pain Episodic pain (eg, familial episodic pain) rs33985936* 3: 38894643 C > T V909I 909 0.165 A pain condition rs78812474** 3: 38950107 G > T R86G 86 0.061 A pain condition rs72869687*** 3: 38847244 G > A T1609I 1609 0.054 A pain condition rs13059805**** 3: 38907980 C > T G481E 481 0.013 A pain condition SFN = small fibre neuropathy or pain associated with small fibre neuropathy, SFN with large fibre involvement ¹According to Ensembl, dbSNP *rs33985936 is present in humans at an average cumulative frequency of 17% according to the 1000 Genomes Phase I database, but is higher in AMR, EUR, MXL, PUR, CEU, FIN, GBR, IBS and TSI populations; thus in an embodiment, in the present invention involving Nav1.9 the human is of AMR ancestry, eg, of MXL or PUR ancestry; or of EUR ancestry, eg, of CEU, FIN, GBR, IBS or TSI ancestry. **rs78812474 is present in humans at an average cumulative frequency of 6% according to the 1000 Genomes Phase I database, but is higher in AFR, LWK, YRI, MXL and GBR populations; thus in an embodiment, in the present invention involving Nav1.9 the human is of AFR ancestry, eg, of LWK or YRI ancestry; or of MXL or GBR ancestry. ***rs72869687 is present in humans at an average cumulative frequency of 5% according to the 1000 Genomes Phase I database, but is higher in AFR, ASN, ASW, LWK, YRI, CHB, CHS and JPT populations; thus in an embodiment, in the present invention involving Nav1.9 the human is of AFR ancestry, eg, of ASW, LWK or YRI ancestry; or of ASN ancestry, eg, of CHB, CHS or JPT ancestry. ****rs13059805 is present in humans at an average cumulative frequency of 1% according to the 1000 Genomes Phase I database, but is higher in AMR, EUR, CLM, MXL, CEU, FIN and GBR populations; thus in an embodiment, in the present invention involving Nav1.9 the human is of AMR, EUR, CLM, MXL, CEU, FIN or GBR ancestry. In an embodiment, variations are with reference to transcript ENST00000302328 (the sequence of this transcript being incorporated herein in its entirety).

Optionally for any mutation, the condition is a condition listed in Table 26.

In an embodiment, the human comprises a SNP selected from the SNPs in column 1 of Table 26; optionally also the ligand specifically binds a human Nav1.9 encoded by a nucleotide sequence comprising said SNP.

In an embodiment, the human comprises a Nav1.9 amino acid variation selected from the variations in column 4 of Table 26; optionally also the ligand specifically binds a human Nav1.9 comprising said variation.

In an example, the invention provides a method of treating or reducing the risk of a Nav1.9-mediated disease or condition in a human in need thereof, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E. These amino acid variations are found in naturally-occurring Nav1.9 variants in humans.

Said human comprises a nucleotide sequence encoding a Nav1.9 protein comprising said selected amino acid.

Optionally in any embodiment of the Nav1.9 invention said amino acid is selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P and R225C, eg, wherein the condition is a pain condition.

Optionally in any embodiment of the Nav1.9 invention said amino acid is selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P and F1689L, eg, wherein the condition is SFN, numbness (eg, in feet), tingling (eg, in legs), cramps (eg, in hands, feet or legs), joint pain, decreased vibration sensitivity, or abnormal warmth or cold sensation.

Optionally in any embodiment of the Nav1.9 invention said amino acid is selected from the group consisting of A808G and R225C, eg, wherein the condition is a corresponding condition listed in Table 26.

Alternatively, in any embodiment of the Nav1.9 invention the amino acid is encoded by a SNP selected from the group consisting of rs33985936, rs78812474, rs72869687 and rs13059805, eg, wherein the condition is a corresponding condition listed in Table 26.

The disease or condition is, for example, any disease or condition described above in the Example relating to Nav1.7 and the disclosure of such diseases and conditions are hereby also imported into this example to provide for possible combination of the present invention relating to Nav1.9 (eg, for use in one or more claims herein). In one embodiment, the condition is a condition listed in Table 26 or a symptom of such a condition. In an example, the condition is pain or a symptom of pain, eg, a symptom of neuropathic pain. In an example, the condition is selected from the group consisting of paroxysmal pain (eg, of hands and/or feet), hand pain, foot pain, limb pain (eg, leg pain), cramp (eg, foot cramp or leg cramp), tingling (e, foot, hand or leg tingling) and pain associated with clothing contact with the human. In an example, the condition is an abnormal threshold for warmth or cold sensation (eg, in a foot or feet). In an example, the condition is joint pain. In an example, the condition is dry eye sensation, diahrrhoea, urinary condition, heart palpitations or dizziness. In an example, the condition is insensitivity to pain. In an example, the condition is episodic pain (eg, familial episodic pain).

In one embodiment, the pain is chronic pain.

In one embodiment, the pain is neuropathic pain, eg, chronic neuropathic pain, eg, peripheral neuropathic pain. For example, the pain is painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN) or trigeminal neuralgia (TN).

In an example, the pain is spinal cord injury pain, multiple sclerosis pain, phantom limb pain, post-stroke pain, chronic back pain, osteoarthritis pain, cancer-associated pain or HIV-associated pain.

In one embodiment, the pain is inflammatory pain, eg, chronic inflammatory pain.

In an example, the ligand of the invention comprises an anti-human Nav1.9 binding site, wherein the binding site is a human or humanized binding site, eg, the binding site comprises or consists of a human or humanized antibody variable domain or plurality of variable domains (eg, human VH/VL binding site(s)). Additionally or alternatively, the ligand comprises one or more human antibody constant regions (eg, a human antibody CH1, CH2, CH3 (or all of these) or Fc). In an example, the ligand is an antibody that comprises human or humanized variable regions and human constant regions (eg, bearing one or more mutations to enhance or dampen Fc function in a human patient).

An example provides a ligand (eg, an antibody or antibody fragment) for treating or reducing the risk of a Nav1.9-mediated disease or condition in a human in need thereof, the method comprising administering to said human said ligand, wherein the ligand specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E. Said human comprises a nucleotide sequence encoding a Nav1.9 protein comprising said selected amino acid.

In an example, the invention provides a method of targeting Nav1.9 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E. Said human comprises a nucleotide sequence encoding a Nav1.9 protein comprising said selected amino acid. In an example, the human is suffering from or at risk of a Nav1.9-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.9-mediated disease or condition in the human.

An example also provides a ligand (eg, an antibody or antibody fragment) for targeting Nav1.9 in a human, the method comprising administering to said human said ligand, wherein the ligand specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E. Said human comprises a nucleotide sequence encoding a (eg, said) Nav1.9 protein comprising said selected amino acid. In an example, the human is suffering from or at risk of a Nav1.9-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.9-mediated disease or condition in the human.

In an embodiment, (i) the antibody or fragment comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.9 protein comprising said amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

Additionally or alternatively, in an embodiment, (i) the antibody or fragment comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.9 protein comprising said amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an example, the ligand, antibody or fragment is for treating or reducing the risk of (or treats or reduces the risk of) pain or itching, optionally wherein the antibody is a humanized mouse or Camelid (eg, llama or camel) antibody.

In a specific embodiment, the anti-Nav1.9 ligand, antibody or fragment also specifically binds to another Nav selected from Nav1.1-1.8, eg, it specifically binds to Nav1.7 or 1.8.

In a specific embodiment, the anti-Nav1.9 ligand, antibody or fragment of the present invention comprises an Fc region as per the disclosure in the Nav1.7 example above.

The ligand, antibody or fragment according to the invention is for treating or preventing or reducing the risk of (or treats or prevents or reduces the risk of), for example, any disease or condition disclosed in WO2011/051351 or U.S. Pat. No. 7,582,745, the disclosures of which diseases and conditions are incorporated herein by reference for potential inclusion in one or more claims herein. Guidance on obtaining and testing antibodies can also be found in that PCT application.

Further encompassed by the invention is the use of the ligand, antibody or fragment in the manufacture of a medicament for use to attenuate or inhibit a Nav1.9-mediated disease or disorder in a human. Nav1.9-mediated or related disorders which are treated by the ligand, antibody or fragment of the invention include, for example, a pain or itching disease or condition.

Thus, a ligand, antibody or fragment of the invention is useful as a therapeutic agent in the treatment of a condition involving Nav1.9 expression and/or activity. One embodiment, among others, is a method of treatment comprising administering an effective amount of a ligand, antibody or fragment of the invention to a patient in need thereof, wherein functional consequences of Nav1.9 activation are decreased. Another embodiment, among others, is a method of treatment comprising (i) identifying a patient demonstrating Nav1.9 expression or activity, and (ii) administering an effective amount of a ligand, antibody or fragment of the invention to the patient, wherein a functional consequence of Nav1.9 activation are attenuated. An effective amount according to the invention is an amount that modulates (e.g. decreases) the functional consequences of Nav1.9 activation so as to modulate (e.g. decrease or lessen) the severity of at least one symptom of the particular disease or disorder being treated, but not necessarily cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, comprising administering to a patient in need thereof an effective amount of one or more ligands, antibodies or fragments of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein such that the severity of at least one symptom of any of the disorders is reduced. Another embodiment of the invention, among others, is a method of antagonizing at least one effect of Nav1.9 comprising contacting with or administering an effective amount of one or more ligands, antibodies or fragments of the present invention such that said at least one effect of Nav1.9 is antagonized, e.g. the ability of Nav1.9 to form an ion channel, such as a sodium channel.

Tailoring Antibodies to Rarer Nav1.9 Variant Profile

As outlined herein (for example, in the context of PCSK9 in Example 1 or in the context of the Nav1.7 Example herein), the invention includes the possibility to tailor treatment of humans further by selecting antibody-based ligands with variable domains and/or constant domains based on gene segments found in many humans of the ethnic populations where the variant TOI forms are found to meet the selection criteria of the invention. This also applies mutatis mutandis where the TOI is human Nav1.9, as in the present example. Thus, all disclosure herein relating to tailoring variable and/or constant domains apply to the present example, relating to Nav1.9 and is combinable for use in one or more claims herein. All disclosure relating to variable and constant regions and polymorphism under the subtitle “Tailoring Antibodies to Rarer Nav1.7 Variant Profile” in the Nav1.7 Example is also incorporated into the present Nav1.9 Example for use mutatis mutandis with the present Nav1.9 invention and for possible inclusion in Nav1.9-limited claims herein.

Determination of Specific Binding of Ligands of the Invention to NAV1.9 Variants

The specific binding of ligands of the invention to Nav1.9 variants can be performed using the SPR method described in Example 1.

Embodiments are provided as follows:—

Methods with VH Tailoring

-   1. A method of treating or reducing the risk of a Nav1.9-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a human Nav1.9 protein     that comprises an amino acid selected from the group consisting of     I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C,     A808G, V909I, R86G, T1609I and G481E;     -   wherein (i) the ligand comprises a VH domain derived from the         recombination of a human VH segment, a human D gene segment and         a human JH segment, the human VH segment encoding the framework         1 of SEQ ID NO: 40 and wherein said human comprises a VH gene         segment encoding the framework 1 of SEQ ID NO: 40, or the human         expresses VH domains that comprise the framework 1 of SEQ ID NO:         40; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.9 protein comprising said amino acid selected from the         group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,         F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an alternative, clause 1 provides:—

A method of targeting Nav1.9 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E; wherein (i) the ligand comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding the framework 1 of SEQ ID NO: 40 and wherein said human comprises a VH gene segment encoding the framework 1 of SEQ ID NO: 40, or the human expresses VH domains that comprise the framework 1 of SEQ ID NO: 40; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.9 protein comprising said amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an example, the human is suffering from or at risk of a Nav1.9-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.9-mediated disease or condition in the human.

-   2. The method of clause 1, wherein said amino acid is selected from     the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P     and F1689L. -   3. The method of clause 2, wherein the condition is small fibre     neuropathy, numbness, tingling, cramps, joint pain, decreased     vibration sensitivity or abnormal warmth or cold sensation. -   4. The method of clause 1, wherein said amino acid is A808G or     R225C. -   5. The method of any preceding clause, wherein the VH gene segment     comprised by said human is a germline VH gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.9     protein comprising said selected amino acid and/or a Nav1.9 protein     comprising said selected amino acid. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes a Nav1.9 protein comprising said selected amino acid     and/or (b) a Nav1.9 protein comprising said selected amino acid,     optionally, wherein the determining step is performed before     administration of the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.9 protein comprising said selected amino     acid. -   10. The method of clause 9, wherein the assaying comprises     contacting the biological sample with at least one oligonucleotide     probe comprising a sequence of at least 10 contiguous nucleotides of     a nucleotide sequence encoding a Nav1.9 protein comprising said     selected amino acid or comprising an antisense sequence of said     contiguous nucleotides, wherein said sequence of contiguous     nucleotides comprises a nucleotide sequence encoding said selected     amino acid, thereby forming a complex when at least one nucleotide     sequence encoding the Nav1.9 protein comprising said selected amino     acid is present; and detecting the presence or absence of the     complex, wherein detecting the presence of the complex determines     that the human comprises the Nav1.9 protein comprising said selected     amino acid. -   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.9 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 138 or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.9 protein comprising     said selected amino acid. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The method of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The method of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs33985936, rs78812474, rs72869687 and rs13059805. -   21. The method of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is administered by inhaled,     intravenous or subcutaneous administration and/or is comprised in an     inhalable or injectable preparation.

Ligands with VH Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of a Nav1.9-mediated disease     or condition (eg, asthma) in a human in need thereof, wherein the     ligand specifically binds a human Nav1.9 protein that comprises an     amino acid selected from the group consisting of I381T, K419N,     A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I,     R86G, T1609I and G481E;     -   wherein (i) the ligand comprises a VH domain derived from the         recombination of a human VH segment, a human D gene segment and         a human JH segment, the human VH segment encoding the framework         1 of SEQ ID NO: 40 and wherein said human comprises a VH gene         segment encoding the framework 1 of SEQ ID NO: 40, or the human         expresses VH domains that comprise the framework 1 of SEQ ID NO:         40; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.9 protein comprising said amino acid selected from the         group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,         F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting Nav1.9 in a human, wherein the ligand specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E;

-   -   wherein (i) the ligand comprises a VH domain derived from the         recombination of a human VH segment, a human D gene segment and         a human JH segment, the human VH segment encoding the framework         1 of SEQ ID NO: 40 and wherein said human comprises a VH gene         segment encoding the framework 1 of SEQ ID NO: 40, or the human         expresses VH domains that comprise the framework 1 of SEQ ID NO:         40; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.9 protein comprising said amino acid selected from the         group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,         F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an example, the human is suffering from or at risk of a Nav1.9-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.9-mediated disease or condition in the human.

-   2. The ligand of clause 1, wherein said amino acid is selected from     the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P     and F1689L. -   3. The ligand of clause 2, wherein the condition is small fibre     neuropathy, numbness, tingling, cramps, joint pain, decreased     vibration sensitivity or abnormal warmth or cold sensation. -   4. The ligand of clause 1, wherein said amino acid is A808G or     R225C. -   5. The ligand of any preceding clause, wherein the VH gene segment     comprised by said human is a germline VH gene segment. -   6. The ligand of any preceding clause said method comprising, before     said administering, selecting a human comprising said nucleotide     sequence of (ii), wherein the human is the human of clause 1. -   7. The ligand of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.9     protein comprising said selected amino acid selected and/or a Nav1.9     protein comprising said selected amino acid. -   8. The ligand of any preceding clause, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes a Nav1.9 protein comprising said selected     amino acid and/or (b) a Nav1.9 protein comprising said selected     amino acid, optionally, wherein the determining step is performed     before administration of the antibody to the human. -   9. The ligand of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.9 protein comprising said selected amino     acid. -   10. The ligand of clause 9, wherein the assaying comprises     -   contacting the biological sample with at least one         oligonucleotide probe comprising a sequence of at least 10         contiguous nucleotides of a nucleotide sequence encoding a         Nav1.9 protein comprising said selected amino acid or comprising         an antisense sequence of said contiguous nucleotides, wherein         said sequence of contiguous nucleotides comprises a nucleotide         sequence encoding said selected amino acid, thereby forming a         complex when at least one nucleotide sequence encoding the         Nav1.9 protein comprising said selected amino acid is present;         and detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the Nav1.9 protein comprising said selected amino         acid. -   11. The ligand of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The ligand of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.9 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 138, or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.9 protein comprising     said selected amino acid. -   14. The ligand of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The ligand of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The ligand of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The ligand of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The ligand of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The ligand of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The ligand of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs33985936, rs78812474, rs72869687 and rs13059805. -   21. The ligand of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is for inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation.

Methods with Gamma-4 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.9-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a human Nav1.9 protein     that comprises an amino acid selected from the group consisting of     I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C,     A808G, V909I, R86G, T1609I and G481E;     -   wherein (i) the ligand comprises a human gamma-4 heavy chain         constant region that comprises a Leu at position 189 shown in         SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73         and wherein said human comprises (i) an IGHG4*01 human heavy         chain constant region gene segment, or the human expresses         antibodies comprising human gamma-4 heavy chain constant regions         comprising a Len at position 189 shown in SEQ ID NO: 73 or an         Arg at position 289 shown in SEQ ID NO: 73; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.9 protein comprising said amino acid selected from the         group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,         F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an alternative, clause 1 provides:—

A method of targeting Nav1.9 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E;

wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.9 protein comprising said amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an example, the human is suffering from or at risk of a Nav1.9-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.9-mediated disease or condition in the human.

-   2. The method of clause 1, wherein said amino acid is selected from     the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P     and F1689L. -   3. The method of clause 2, wherein the condition is small fibre     neuropathy, numbness, tingling, cramps, joint pain, decreased     vibration sensitivity or abnormal warmth or cold sensation. -   4. The method of clause 1, wherein said amino acid is A808G or     R225C. -   5. The method of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.9     protein comprising said selected amino acid and/or a Nav1.9 protein     comprising said selected amino acid. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes a Nav1.9 protein comprising said selected amino acid     and/or (b) a Nav1.9 protein comprising said selected amino acid,     optionally, wherein the determining step is performed before     administration of the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.9 protein comprising said selected amino     acid. -   10. The method of clause 9, wherein the assaying comprises     -   contacting the biological sample with at least one         oligonucleotide probe comprising a sequence of at least 10         contiguous nucleotides of a nucleotide sequence encoding a         Nav1.9 protein comprising said selected amino acid or comprising         an antisense sequence of said contiguous nucleotides, wherein         said sequence of contiguous nucleotides comprises a nucleotide         sequence encoding said selected amino acid, thereby forming a         complex when at least one nucleotide sequence encoding the         Nav1.9 protein comprising said selected amino acid is present;         and detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the Nav1.9 protein comprising said selected amino         acid. -   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.9 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 138 or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.9 protein comprising     said selected amino acid. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The method of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The method of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs33985936, rs78812474, rs72869687 and rs13059805. -   21. The method of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is administered by inhaled,     intravenous or subcutaneous administration and/or is comprised in an     inhalable or injectable preparation. -   22. The method of any preceding clause, wherein the human gamma-4     heavy chain constant region of the ligand comprises the amino acid     sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof. -   23. The method of any preceding clause, wherein the human gamma-4     heavy chain constant region comprises 228P and 235E.

Ligands with Gamma-4 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of a Nav1.9-mediated disease     or condition (eg, pain) in a human in need thereof, wherein the     ligand specifically binds a human Nav1.9 protein that comprises an     amino acid selected from the group consisting of I381T, K419N,     A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I,     R86G, T1609I and G481E;     -   wherein (i) the ligand comprises a human gamma-4 heavy chain         constant region that comprises a Leu at position 189 shown in         SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73         and wherein said human comprises (i) an IGHG4*01 human heavy         chain constant region gene segment, or the human expresses         antibodies comprising human gamma-4 heavy chain constant regions         comprising a Leu at position 189 shown in SEQ ID NO: 73 or an         Arg at position 289 shown in SEQ ID NO: 73; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.9 protein comprising said amino acid selected from the         group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,         F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting Nav1.9 in a human, wherein the ligand specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E;

wherein (i) the ligand comprises a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-4 heavy chain constant regions comprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.9 protein comprising said amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an example, the human is suffering from or at risk of a Nav1.9-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.9-mediated disease or condition in the human.

-   2. The ligand of clause 1, wherein said amino acid is selected from     the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P     and F1689L. -   3. The ligand of clause 2, wherein the condition is small fibre     neuropathy, numbness, tingling, cramps, joint pain, decreased     vibration sensitivity or abnormal warmth or cold sensation. -   4. The ligand of clause 1, wherein said amino acid is A808G or     R225C. -   5. The ligand of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The ligand of any preceding clause said method comprising, before     said administering, selecting a human comprising said nucleotide     sequence of (ii), wherein the human is the human of clause 1. -   7. The ligand of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.9     protein comprising said selected amino acid selected and/or a Nav1.9     protein comprising said selected amino acid. -   8. The ligand of any preceding clause, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes a Nav1.9 protein comprising said selected     amino acid and/or (b) a Nav1.9 protein comprising said selected     amino acid, optionally, wherein the determining step is performed     before administration of the antibody to the human. -   9. The ligand of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.9 protein comprising said selected amino     acid. -   10. The ligand of clause 9, wherein the assaying comprises     -   contacting the biological sample with at least one         oligonucleotide probe comprising a sequence of at least 10         contiguous nucleotides of a nucleotide sequence encoding a         Nav1.9 protein comprising said selected amino acid or comprising         an antisense sequence of said contiguous nucleotides, wherein         said sequence of contiguous nucleotides comprises a nucleotide         sequence encoding said selected amino acid, thereby forming a         complex when at least one nucleotide sequence encoding the         Nav1.9 protein comprising said selected amino acid is present;         and     -   detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the Nav1.9 protein comprising said selected amino         acid. -   11. The ligand of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The ligand of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.9 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 138, or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.9 protein comprising     said selected amino acid. -   14. The ligand of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The ligand of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The ligand of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The ligand of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The ligand of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The ligand of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The ligand of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs33985936, rs78812474, rs72869687 and rs13059805. -   21. The ligand of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is for inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation. -   22. The ligand of any preceding clause, wherein the human gamma-4     heavy chain constant region of the ligand comprises the amino acid     sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof. -   23. The ligand of any preceding clause, wherein the human gamma-4     heavy chain constant region comprises 228P and 235E.

Methods with Gamma-2 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.9-mediated     disease or condition in a human in need thereof, the method     comprising administering to said human a ligand (eg, an antibody or     antibody fragment) that specifically binds a human Nav1.9 protein     that comprises an amino acid selected from the group consisting of     I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C,     A808G, V909I, R86G, T1609I and G481E;     -   wherein (i) the ligand comprises a human gamma-2 heavy chain         constant region that comprises an amino acid selected from the         group consisting of a Pro at position 72 shown in SEQ ID NO: 6,         an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position         76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID         NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and         wherein said human comprises (i) an IGHG2*01 human heavy chain         constant region gene segment, or the human expresses antibodies         comprising human gamma-2 heavy chain constant regions comprising         said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at         position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in         SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala         at position 257 shown in SEQ ID NO: 6; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.9 protein comprising said amino acid selected from the         group consisting of I138T, K419N, A582T, A681D, A842P, L1158P,         F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an alternative, clause 1 provides:—

A method of targeting Nav1.9 in a human, the method comprising administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E;

wherein (i) the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.9 protein comprising said amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an example, the human is suffering from or at risk of a Nav1.9-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.9-mediated disease or condition in the human.

-   2. The method of clause 1, wherein said amino acid is selected from     the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P     and F1689L. -   3. The method of clause 2, wherein the condition is small fibre     neuropathy, numbness, tingling, cramps, joint pain, decreased     vibration sensitivity or abnormal warmth or cold sensation. -   4. The method of clause 1, wherein said amino acid is A808G or     R225C. -   5. The method of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The method of any preceding clause comprising, before said     administering, selecting a human comprising said nucleotide sequence     of (ii), wherein the human is the human of clause 1. -   7. The method of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.9     protein comprising said selected amino acid and/or a Nav1.9 protein     comprising said selected amino acid. -   8. The method of any preceding clause, comprising the step of     determining that the human comprises (a) the nucleotide sequence     that encodes a Nav1.9 protein comprising said selected amino acid     and/or (b) a Nav1.9 protein comprising said selected amino acid,     optionally, wherein the determining step is performed before     administration of the antibody to the human. -   9. The method of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.9 protein comprising said selected amino     acid. -   10. The method of clause 9, wherein the assaying comprises     -   contacting the biological sample with at least one         oligonucleotide probe comprising a sequence of at least 10         contiguous nucleotides of a nucleotide sequence encoding a         Nav1.9 protein comprising said selected amino acid or comprising         an antisense sequence of said contiguous nucleotides, wherein         said sequence of contiguous nucleotides comprises a nucleotide         sequence encoding said selected amino acid, thereby forming a         complex when at least one nucleotide sequence encoding the         Nav1.9 protein comprising said selected amino acid is present;         and     -   detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the Nav1.9 protein comprising said selected amino         acid. -   11. The method of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The method of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The method of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.9 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 138 or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.9 protein comprising     said selected amino acid. -   14. The method of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The method of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The method of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The method of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The method of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The method of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The method of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs33985936, rs78812474, rs72869687 and rs13059805. -   21. The method of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is administered by inhaled,     intravenous or subcutaneous administration and/or is comprised in an     inhalable or injectable preparation. -   22. The method of any preceding clause, wherein the human gamma-2     heavy chain constant region of the ligand comprises IGHG2*01 amino     acid sequence or an ADCC inactivated version thereof.

Ligands with Gamma-2 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a     method of treating or reducing the risk of a Nav1.9-mediated disease     or condition (eg, pain) in a human in need thereof, wherein the     ligand specifically binds a human Nav1.9 protein that comprises an     amino acid selected from the group consisting of I381T, K419N,     A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I,     R86G, T1609I and G481E;     -   wherein (i) the ligand comprises a human gamma-2 heavy chain         constant region that comprises an amino acid selected from the         group consisting of a Pro at position 72 shown in SEQ ID NO: 6,         an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position         76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID         NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and         wherein said human comprises (i) an IGHG2*01 human heavy chain         constant region gene segment, or the human expresses antibodies         comprising human gamma-2 heavy chain constant regions comprising         said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at         position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in         SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala         at position 257 shown in SEQ ID NO: 6; and     -   wherein (ii) said human comprises a nucleotide sequence encoding         said Nav1.9 protein comprising said amino acid selected from the         group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,         F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an alternative, paragraph 1 provides:—

A ligand (eg, an antibody or antibody fragment) for use in a method of targeting Nav1.9 in a human, wherein the ligand specifically binds a human Nav1.9 protein that comprises an amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E;

wherein (i) the ligand comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6; and

wherein (ii) said human comprises a nucleotide sequence encoding said Nav1.9 protein comprising said amino acid selected from the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V909I, R86G, T1609I and G481E.

In an example, the human is suffering from or at risk of a Nav1.9-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.9-mediated disease or condition in the human.

-   2. The ligand of clause 1, wherein said amino acid is selected from     the group consisting of J381T, K419N, A582T, A681D, A842P, L1158P     and F1689L. -   3. The ligand of clause 2, wherein the condition is small fibre     neuropathy, numbness, tingling, cramps, joint pain, decreased     vibration sensitivity or abnormal warmth or cold sensation. -   4. The ligand of clause 1, wherein said amino acid is A808G or     R225C. -   5. The ligand of any preceding clause, wherein the constant region     gene segment comprised by said human is a germline gene segment. -   6. The ligand of any preceding clause said method comprising, before     said administering, selecting a human comprising said nucleotide     sequence of (ii), wherein the human is the human of clause 1. -   7. The ligand of any preceding clause, wherein the human has been     determined to comprise the nucleotide sequence that encodes a Nav1.9     protein comprising said selected amino acid selected and/or a Nav1.9     protein comprising said selected amino acid. -   8. The ligand of any preceding clause, said method comprising the     step of determining that the human comprises (a) the nucleotide     sequence that encodes a Nav1.9 protein comprising said selected     amino acid and/or (b) a Nav1.9 protein comprising said selected     amino acid, optionally, wherein the determining step is performed     before administration of the antibody to the human. -   9. The ligand of clause 8, wherein the step of determining comprises     assaying a biological sample from the human for a nucleotide     sequence encoding a Nav1.9 protein comprising said selected amino     acid. -   10. The ligand of clause 9, wherein the assaying comprises     -   contacting the biological sample with at least one         oligonucleotide probe comprising a sequence of at least 10         contiguous nucleotides of a nucleotide sequence encoding a         Nav1.9 protein comprising said selected amino acid or comprising         an antisense sequence of said contiguous nucleotides, wherein         said sequence of contiguous nucleotides comprises a nucleotide         sequence encoding said selected amino acid, thereby forming a         complex when at least one nucleotide sequence encoding the         Nav1.9 protein comprising said selected amino acid is present;         and     -   detecting the presence or absence of the complex, wherein         detecting the presence of the complex determines that the human         comprises the Nav1.9 protein comprising said selected amino         acid. -   11. The ligand of clause 9, wherein the assaying comprises nucleic     acid amplification and optionally one or more methods selected from     sequencing, next generation sequencing, nucleic acid hybridization,     and allele-specific amplification and/or wherein the assaying is     performed in a multiplex format. -   12. The ligand of clause 9 or 11, wherein said biological sample     comprises serum, blood, faeces, tissue, a cell, urine and/or saliva     of said human. -   13. The ligand of any preceding clause, wherein said human is     indicated as heterozygous for a nucleotide sequence encoding the     Nav1.9 protein comprising said selected amino acid, optionally,     wherein said human is further indicated as comprising the nucleotide     sequence of SEQ ID NO: 138, or said human is indicated as homozygous     for a nucleotide sequence encoding the Nav1.9 protein comprising     said selected amino acid. -   14. The ligand of any preceding clause, wherein said human is or has     been further determined to be substantially resistant to a pain or     itching treatment. -   15. The ligand of any preceding clause, wherein said human is     receiving or has received a pain or itching treatment or has reduced     responsiveness to a pain or itching treatment. -   16. The ligand of any preceding clause, wherein said disease or     condition is a pain or itching disease or condition. -   17. The ligand of any preceding clause, wherein said disease or     condition is neuropathic pain. -   18. The ligand of any preceding clause, wherein said human has been     diagnosed with at least one condition recited in clause 16 or 17. -   19. The ligand of any preceding clause, wherein said antibody or     antibody fragment treats or reduces the risk in said human of a     condition recited in clause 16 or 17. -   20. The ligand of any preceding clause, wherein the nucleotide     sequence comprises one or more SNPs selected from the group     consisting of rs33985936, rs78812474, rs72869687 and rs13059805. -   21. The ligand of any preceding clause, wherein said ligand (eg,     antibody or antibody fragment) is for inhaled, intravenous or     subcutaneous administration and/or is comprised in an inhalable or     injectable preparation. -   22. The ligand of any preceding clause, wherein the human gamma-2     heavy chain constant region of the ligand comprises IGHG2*01 amino     acid sequence or an ADCC inactivated version thereof.

In any embodiment or example of the Nav1.9-focused invention, the method optionally reduces a symptom of neuropathic pain or inflammatory pain; or optionally the ligand is for reducing a symptom of neuropathic pain or inflammatory pain.

In any embodiment or example of the Nav1.9-focused invention said selected amino acid may be regarded as a mutation in SEQ ID NO: 137.

In an example, the human is suffering from or at risk of a Nav1.9-mediated disease or condition. In an example, the method treats or reduces the risk of a Nav1.9-mediated disease or condition in the human. 

We claim:
 1. A method of increasing erythopoiesis in a human subject, the method comprising administering a trap, and antibody or an antibody fragment that binds a human TMPRSS6 encoded by a TMPRSS6 nucleotide sequence comprising a SNP selected from the group consisting of: rs855791, rs2543519, rs2235324 and rs1421312; wherein the trap, antibody or antibody fragment comprises a constant region selected from the group consisting of: (a) a human gamma-4 heavy chain constant region that comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (b) a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42; and wherein (i) the human subject comprises a TMPRSS6 nucleotide sequence comprising said selected SNP; and (ii) the human subject comprises a constant region gene segment encoding said selected constant region; or the human subject expresses antibodies comprising said selected constant region.
 2. The method of claim 1, wherein the human subject is homozygous for said selected SNP.
 3. The method of claim 1, wherein the trap, antibody or antibody fragment comprises an IGHG1*01 human heavy chain constant region.
 4. The method of claim 1, wherein the trap, antibody or antibody fragment comprises an IGHG4*01 human heavy chain constant region.
 5. The method of claim 1, comprising, before said administering, selecting a human subject comprising said TMPRSS6 nucleotide sequence, wherein the human is the human of claim
 1. 6. The method of claim 1, wherein the human subject has been determined to comprise said TMPRSS6 nucleotide sequence.
 7. The method of claim 1, comprising the step of determining that the human subject comprises said TMPRSS6 nucleotide sequence, optionally, wherein the determining step is performed before administration of the trap, antibody, or antibody fragment to the human.
 8. The method of claim 7, wherein the step of determining comprises assaying a biological sample from the human subject for a nucleotide sequence encoding said SNP.
 9. The method of claim 8, wherein the assaying comprises contacting the biological sample with at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides that can specifically hybridize to and identify in the biological sample a nucleotide sequence comprising said SNP or that specifically hybridizes to an antisense of said sequence, wherein said nucleic acid hybridizes to said SNP or hybridizes to an antisense sequence thereby forming a complex when at least one nucleotide sequence comprising said SNP is present; and/or at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides of a nucleotide sequence comprising said SNP or comprising an antisense sequence of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises said SNP thereby forming a complex when the nucleotide sequence comprising said SNP is present; and detecting the presence or absence of the complex, wherein detecting the presence of the complex determines that the human subject comprises a nucleotide sequence encoding TMPRSS6 and comprising said SNP.
 10. The method of claim 8, wherein the assaying comprises nucleic acid amplification and optionally one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification or wherein the assaying is performed in a multiplex format.
 11. The method of claim 8, wherein said biological sample comprises serum, blood, faeces, tissue, a cell, urine or saliva of said human subject.
 12. The method of claim 1, wherein said human subject is or has been further determined to be substantially resistant to an anaemia treatment.
 13. The method of claim 1, wherein said human subject is receiving or has received an anaemia treatment or has reduced responsiveness to an anaemia treatment.
 14. The method of claim 1, wherein said trap, antibody or antibody fragment is administered by intravenous or subcutaneous injection or is comprised in an injectable preparation.
 15. The method of claim 1, wherein the trap, antibody or antibody fragment is a human trap, antibody, or antibody fragment.
 16. The method of claim 1, wherein said human subject is or has been further determined to be substantially resistant to an EPO anaemia treatment.
 17. The method of claim 1, wherein said human subject is receiving or has received an EPO anaemia treatment or has reduced responsiveness to an EPO anaemia treatment. 